A Critical Evaluation of The Biological Construct

PERSPECTIVE

published: 12 March 2019

doi: 10.3389/fphys.2019.00247
A Critical Evaluation of the Biological

Construct Skeletal Muscle
Hypertrophy: Size Matters but So
Does the Measurement
Cody T. Haun 1* , Christopher G. Vann 2 , Brandon M. Roberts 3 , Andrew D. Vigotsky 4 ,
Brad J. Schoenfeld 5 and Michael D. Roberts 2*
1
Department of Exercise Science, LaGrange College, LaGrange, GA, United States, 2 School of Kinesiology, Auburn
University, Auburn, AL, United States, 3 Department of Cell, Developmental and Integrative Biology, University of Alabama
at Birmingham, Birmingham, AL, United States, 4 Department of Biomedical Engineering, Northwestern University, Evanston,
IL, United States, 5 Department of Health Sciences, CUNY Lehman College, Bronx, NY, United States
Skeletal muscle is highly adaptable and has consistently been shown to morphologically
respond to exercise training. Skeletal muscle growth during periods of resistance
training has traditionally been referred to as skeletal muscle hypertrophy, and this
Edited by: manifests as increases in muscle mass, muscle thickness, muscle area, muscle
Peter Michael Lalley, volume, and muscle fiber cross-sectional area (fCSA). Delicate electron microscopy
University of Wisconsin School
of Medicine and Public Health,
and biochemical techniques have also been used to demonstrate that resistance
United States exercise promotes ultrastructural adaptations within muscle fibers. Decades of research
Reviewed by: in this area of exercise physiology have promulgated a widespread hypothetical
Thomas Chaillou,
model of training-induced skeletal muscle hypertrophy; specifically, fCSA increases are
Örebro University, Sweden
Paul Timothy Reidy, accompanied by proportional increases in myofibrillar protein, leading to an expansion
The University of Utah, United States in the number of sarcomeres in parallel and/or an increase in myofibril number. However,
*Correspondence: there is ample evidence to suggest that myofibrillar protein concentration may be diluted
Cody T. Haun
[email protected]
through sarcoplasmic expansion as fCSA increases occur. Furthermore, and perhaps
Michael D. Roberts more problematic, are numerous investigations reporting that pre-to-post training
[email protected]
change scores in macroscopic, microscopic, and molecular variables supporting
Specialty section:
this model are often poorly associated with one another. The current review first
This article was submitted to provides a brief description of skeletal muscle composition and structure. We then
Exercise Physiology,
provide a historical overview of muscle hypertrophy assessment. Next, current-day
a section of the journal
Frontiers in Physiology methods commonly used to assess skeletal muscle hypertrophy at the biochemical,
Received: 28 November 2018 ultramicroscopic, microscopic, macroscopic, and whole-body levels in response to
Accepted: 25 February 2019 training are examined. Data from our laboratory, and others, demonstrating correlations
Published: 12 March 2019
(or the lack thereof) between these variables are also presented, and reasons for
Citation:
Haun CT, Vann CG, Roberts BM,
comparative discrepancies are discussed with particular attention directed to studies
Vigotsky AD, Schoenfeld BJ and reporting ultrastructural and muscle protein concentration alterations. Finally, we critically
Roberts MD (2019) A Critical
evaluate the biological construct of skeletal muscle hypertrophy, propose potential
Evaluation of the Biological Construct
Skeletal Muscle Hypertrophy: Size operational definitions, and provide suggestions for consideration in hopes of guiding
Matters but So Does future research in this area.
the Measurement.
Front. Physiol. 10:247. Keywords: myofibrillar protein, sarcoplasmic protein, fiber cross-sectional area, ultrasound, dual x-ray
doi: 10.3389/fphys.2019.00247 absorptiometry, muscle hypertrophy, resistance exercise, skeletal muscle
Frontiers in Physiology | www.frontiersin.org 1 March 2019 | Volume 10 | Article 247

Haun et al. What Is Hypertrophy?
INTRODUCTION in sarcoplasmic volume [i.e., muscle intracellular fluid (ICF)],

rather than an increase in muscle fiber or myofibril number.
Etymology of the term hypertrophy reveals derivation from Likewise, decades later, Goldspink reported that 5 weeks of
the English term “hyper-,” denoting “above” or “beyond,” and resistance-like training of the biceps muscles in mice produced a
Greek term “-trophia,” denoting “growth” or “nourishment.” In 30% increase in fCSA relative to untrained, age-matched controls,
the context of resistance training, skeletal muscle hypertrophy whereas muscle weights in both groups were nearly identical
has been generally defined as an increase in muscle mass and (Goldspink, 1964). Other historical reports of biochemical and
cross-sectional area (CSA) at the whole tissue and cellular ultrastructural changes underpinning changes in muscle size have
levels (Russell et al., 2000). Historically, muscle hypertrophy has been inconsistent, which are discussed in following sections.
been posited to occur in response to the accrual of contractile The aforementioned evidence calls into question what is
or structural proteins due to an increase in the number of meant by the term skeletal muscle hypertrophy. We feel a critical
sarcomeres in parallel in pre-existing myofibrils of muscle evaluation of the term and the construct validity of assessments
fibers, which results in an increase in fiber cross-sectional employed warrant special consideration by researchers moving
area (fCSA) (Russell et al., 2000). Accordingly, it is logical to forward in this area of inquiry. In particular, researchers invoking
assume that investigations reporting increases in muscle size the term should agree on an operational definition so that
and myofibrillar protein alterations would clearly demonstrate the construct validity of an assessment or assessments can be
this phenomenon. However, while skeletal muscle hypertrophy better characterized and adopted for future research endeavors.
is considered a hallmark adaptation of resistance training of Therefore, we begin this review by briefly describing skeletal
sufficient duration, there have been inconsistent observations in muscle composition and structure, and provide a historical
the scientific literature dependent upon the outcome variables overview of the scientific assessment of muscle hypertrophy.
being reported. This, in part, is likely due to numerous methods Next, we discuss laboratory-based measurements used to assess
being used to assess skeletal muscle hypertrophy, and each of training-induced skeletal muscle hypertrophy at the biochemical,
these methods assess distinct characteristics of skeletal muscle. ultrastructural, histological, and gross anatomical levels, and
Many studies in the literature detect regional adaptations using highlight the strengths and limitations of these approaches
dual-energy x-ray absorptiometry (DXA), computed tomography as well as how they differ from one another. We then
(CT) scanning, magnetic resonance imaging (MRI), and/or present whole-body, whole-tissue, microscopic, and biochemical
ultrasound assessment. Additionally, the percutaneous muscle assessments of skeletal muscle hypertrophy obtained by our
biopsy technique has allowed exercise scientists to examine laboratory, and others’, which demonstrate the degree of
pre- and post-intervention differences in muscle fCSA. While agreeability – or lack thereof – between methods with particular
these techniques have provided excellent insight regarding how attention directed to muscle protein concentration alterations.
exercise training affects body composition and muscle tissue Finally, we provide potential operational definitions, suggestions
morphology, recent work from our laboratories, and that of for future research, and discuss methods that could be adopted to
others, suggest that pre- to post-training change scores in these more accurately assess the biological construct of skeletal muscle
measurements often correlate poorly with one another. hypertrophy. While this review is centered on empirical data
We are not the first to propose that measures and scales of obtained in humans, reference to pertinent animal models are
muscle growth are not strongly related. For instance, Dr. Edward also provided where applicable given that this area of human
E. Gordon published a commentary by Gordon (1967) stating: physiology has been largely preceded by delicate animal work.
First, what is meant by hypertrophy: increase in girth of a limb,

volume of a muscle, or the related weight? Or, are we referring SKELETAL MUSCLE COMPOSITION AND
to the individual muscle fiber, the smallest anatomical unit of STRUCTURE
muscle? In [hypertrophy], the gross and microscopic dimensions
are regarded as running parallel courses, and therefore, as Skeletal muscle tissue can be categorized into the following
being interchangeable. There would be no serious error if in levels of organization: (a) whole muscle sheathed by fascia
hypertrophy the sum of the enlarged parts equalled an increased (i.e., epimysium), (b) muscle fibers within fascicle bundles
whole. But such an equation is not always found. (p. 129) (i.e., peri- and endomysium), (c) myofibrils within individual
muscle fibers, (d) sarcomeres within individual myofibrils, and
Gordon went on to note these discrepancies in his own (e) proteins (e.g., actin, myosin, and titin) within individual
research and in the work from some of his contemporaries, sarcomeres (Figure 1).
stating that there appears to be “a complete dissociation between Whole skeletal muscle is sheathed with connective tissue,
whole muscle and fiber size in trained animals” (p. 130). Indeed, primarily composed of collagen protein, and is ∼75% fluid
historical literature is rife with examples that further support this by volume (Kjaer, 2004). Skeletal muscle can be separated
narrative. For example, in the first formal study on work-induced into intracellular (i.e., beneath the muscle fiber membrane)
hypertrophy, Morpurgo reported 26% greater fCSA values in and extracellular (i.e., outside the muscle fiber membrane)
run-trained versus untrained animals, although only a 13% components. The extracellular component is primarily composed
increase in whole-muscle CSA was observed in the former group of connective tissue and vasculature, and connective tissue
(Morpurgo, 1897). Morpurgo related this finding to an increase generally occupies between ∼1–20% of human skeletal muscle

FIGURE 1 | Hierarchical structure of muscle. Pictured is the hierarchical structure of muscle described in text.
which further separates muscle into fascicular bundles of

fibers and individual muscle fibers (Kjaer, 2004). Notably, the
connective tissue component is also adaptable and can vary in its
contribution to skeletal muscle size and strength. Muscle fibers
consist primarily of myofibrils, mitochondria, and a specialized
structure known as the sarcoplasmic reticulum. These are the
three major components of muscle fibers (Lindstedt et al., 1998),
although glycogen also constitutes ∼2–3% and intramuscular
triglycerides ∼5% on average (van Loon et al., 2003; Gallagher
et al., 2005). The human skeletal muscle proteome was recently
“reappraised” by Gonzalez-Freire et al. (2017), who employed
sensitive mass spectrometry-based techniques. Based on this
analysis, most of the proteins in skeletal muscle, by percentage,
are involved in metabolic processes rather contraction directly.
This counters the assumption that most of the proteins in
skeletal muscle serve a direct contractile role. The authors also
categorized around 40% of the total number of proteins in skeletal
muscle as enzymes and under 10% as contractile. Furthermore,
∼20% of proteins were characterized as mitochondrial apparently
serving roles in oxidative metabolism. Notably, these percentages FIGURE 2 | Composition of skeletal muscle tissue. These composition
estimates are based upon numerous studies which have utilized biochemical
are relative to the total number of proteins in human skeletal and proteomics-based assessments described in text. IMTG, intramuscular
muscle, and not the concentration of proteins within skeletal triglycerides; EC, extracellular; IC, intracellular; MF, myofibrillar; SARCO,
muscle. Traditionally, ∼60–70% of the human skeletal muscle sarcoplasmic; MITO, mitochondrial.
mixed protein pool has been characterized as myofibrillar,
∼20–30% as sarcoplasmic, and ∼5–10% as mitochondrial (Haus
et al., 2007). Other estimates suggest that myosin represents Considering the composition and organization of skeletal
∼50% of myofibrillar protein concentration and actin ∼20% muscle tissue, it seems logical that training-induced increases
(Yates and Greaser, 1983; Ingalls et al., 1998). Based on data in fCSA would result in proportional increases in myofibrillar
from Wang (1982) and Yates and Greaser (1983), titin typically protein abundance where concentrations would be largely
represents ∼10% of myofibrillar protein while nebulin, troponin, preserved. Indeed, since ∼60–70% of muscle protein is made
and tropomyosin each represent ∼5%. Quantitatively, these up of myofibrillar proteins, a number of authors have posited
proteins seem to represent ∼95% of all myofibrillar proteins that skeletal muscle hypertrophy in response to resistance
by concentration. Mitochondrial, sarcoplasmic reticulum, and training is due to an increase in myofibrillar protein abundance
t-tubule proteins have been estimated to occupy most of the and an increase in the number of sarcomeres in parallel in
remaining mixed protein pool, while glycolytic enzymes and existent myofibrils (e.g., sarcomerogenesis) or newly synthesized
other protein constituents of the sarcoplasm predominate the myofibrils of existent muscle fibers (e.g., myofibrillogenesis)
remaining pool (Hoppeler and Lindstedt, 1985; Al-Qusairi and (Paul and Rosenthal, 2002; Schoenfeld, 2010; Wisdom et al., 2015;
Laporte, 2011). Figure 2 summarizes the percentage breakdown Franchi et al., 2017). For example, Damas et al. (2018) recently
of these components within muscle fibers. defined “true” hypertrophy as an “. . . accumulation of contractile

and structural muscle proteins adding sarcomeres in parallel to training. Interestingly, all three modes of training reduced the
muscle fibers” (p. 487). However, this mode of skeletal muscle concentration of “myosin fibers,” which Penman characterized as
hypertrophy in response to resistance training has strikingly little the number of myofibrils within a 5 µm2 area of muscle fibers.
direct supportive evidence in human skeletal muscle samples. To In a subsequent 1970 exercise training investigation, Penman
the contrary, select evidence suggests a dilution of myofibrillar reported ∼40% increases in maximum strength, although he also
protein in response to short-term resistance training which is observed reductions in the distance between myosin filaments
described in later sections. To better understand how we have and modest reductions in gross cell diameter following 10 weeks
arrived at the current hypothesized model of training-induced of training which involved 5 sessions per week of both leg
hypertrophy, the following section provides a brief historical extensions and running exercise (Penman, 1970). Penman
overview of the assessment of skeletal muscle hypertrophy. interpreted these findings to indicate that increased strength of
Thenceforth, a survey of current-day methods and more detailed skeletal muscle in response to resistance training without an
discussion of the biological construct of muscle hypertrophy and increase in fiber size involved an increased “packing density”
future directions follows. of contractile elements. Although pioneering, Penman’s 1969
investigation included only 6 subjects (2 in each condition), and 3
subjects in the 1970 investigation, prohibiting sufficient statistical
HISTORICAL ASSESSMENT OF power for population-wide inference. Following Penman’s work,
SKELETAL MUSCLE HYPERTROPHY a seminal 1982 paper by MacDougall et al. (1982) used
transmission electron microscopy (TEM) methods and reported
Morpurgo (1897) was the first researcher to experimentally reductions in biceps brachii myofibrillar and mitochondrial
observe work-induced skeletal muscle hypertrophy in dog volumes as well as an increase in sarcoplasmic volume in response
sartorius muscle following 2 months of run training, which he to 6 months of resistance training in previously untrained human
described as an increase in fiber diameter due to an increase subjects. Furthermore, muscle samples from a group of seven
in sarcoplasmic volume. Following Morpurgo’s seminal work, bodybuilders and powerlifters were compared to the previously
alternative definitions and modes of work-induced skeletal untrained subjects who underwent 6 months of resistance
muscle hypertrophy emerged. For example, Helander (1961) training, and authors reported that this analysis revealed lower
reported greater increases in muscle weights and myofibrillar myofibrillar volume and greater sarcoplasmic volume in the
density in guinea pigs following regimented run training fibers of bodybuilder and powerlifter subjects. These authors
performed 6 days per week for ∼4 months compared to control provocatively concluded that decrements in myofibrillar volume
or restricted-activity animals. Helander concluded, “Exercise with increased resistance training experience may have been
thus enhances the myofilamental density in the muscle cell. . .” related to greater glycogen accumulation and/or increases in
(p. 482), whereas restricted activity “. . . reduces its myofilamental ICF beyond that related to glycogen accumulation (e.g., ∼3 g
density and instead increases sarcoplasmic content” (p. 482). of water/1 g of stored glycogen, increased ion or organelle
Goldspink (1964) published a report that involved training mice abundance), particularly in the bodybuilder and powerlifter
through a pulley apparatus designed to tax the biceps brachii subjects. Notwithstanding, the contribution of sarcoplasmic
muscle. Following 25 days of training, mice were euthanized constituents to muscle fiber hypertrophy during resistance
and muscle was prepared for the histological assessment of training has remained largely unexplored.
muscle fiber size and myofibril number per fiber in cross section. Between 1970 and 1990, studies by Gollnick, Saltin, Tesch,
The author noted a very strong correlation between myofibril Staron, Sale, MacDougall, Alway, and others utilized the
number per fiber and muscle fiber size, and this finding – Bergstrom technique to histologically evaluate muscle fiber
along with Helander’s – helped shape the current-day consensus type composition and fCSA differences between well-trained
that increases in fCSA and myofibrillar protein accretion are weightlifters and untrained subjects (Gollnick et al., 1972; Staron
proportional during resistance training. Other animal trials in et al., 1984; Tesch et al., 1984; Larsson and Tesch, 1986; Sale
the 20th century agreed with or refuted these findings (Gordon et al., 1987; Alway et al., 1988). While most of these studies
et al., 1967; Seiden, 1976), and Goldspink and Howells (1974) examined the vastus lateralis (VL), some studies biopsied the
associated disparate findings on specific modes of hypertrophy deltoid, soleus, biceps brachii, or trapezius muscles. In the 1990s,
with large variations in experimental design, and specifically, several exercise physiology laboratories sought to determine how
differences in the dose of exercise. weeks to months of resistance training affected VL muscle fCSA
The introduction of the Duchenne biopsy needle allowed for values in untrained individuals. Notably, Staron, Hikida, and
percutaneous open biopsies to be obtained from humans (Parent, many others performed seminal work in this area (Staron et al.,
2005). While isolated papers in the 1950s reported the allocation 1990, 1991, 1994; Wang et al., 1993), and this research has
of a biopsy technique in humans, it was not until Jonas Bergstrom been carried on by several other laboratories in the 21st century
introduced his needle sampling technique in 1962 that these (Hikida et al., 2000; Kadi et al., 2004; Kim et al., 2005; Mackey
methods were utilized more openly in human research related et al., 2007, 2011; Petrella et al., 2008; Mitchell et al., 2012;
to skeletal muscle hypertrophy (Ekblom, 2017). Penman (1969) Snijders et al., 2016; Stec et al., 2016; Reidy et al., 2017b; Mobley
was the first to characterize ultrastructural alterations in human et al., 2018). Collectively, most of these studies have demonstrated
skeletal muscle tissue in response to three forms of exercise that, in general, weeks to months of resistance training increases
training which were categorized as isotonic, isometric, or run mean (type I and II) fCSA. While there have been a handful

of training studies that have integrated both histological and (i.e., myofibrillogenesis). Paul and Rosenthal (2002) eloquently
TEM methods to describe microscopic and ultrastructural summarized this model by stating:
adaptations (Luthi et al., 1986; Toth et al., 2012), most of the
post-1980s research in this area only performed histological “. . . muscle fibers may increase in diameter, as is found in singly
assessments given that TEM methodologies are painstaking and innervated muscles, to increase the number of myofibrils in
not widely accessible. Comparatively fewer investigations have parallel. Alternatively, intrafascicularly terminating fibers could
used biochemical methods to assess skeletal muscle myofibrillar elongate, effectively increasing the number of fibers as well as
and/or sarcoplasmic protein concentration adaptations following myofibrils in parallel” (p. 751).
periods of training, and these studies are discussed below in
greater detail. In addition to the seminal microscopy work In reference to sarcomerogenesis, Wisdom et al. (2015)
discussed above, early post-spaceflight investigations of muscle posited, “On the subcellular scale, in response to elevated forces,
mass and thin filament density changes from LeBlanc et al. more sarcomeres, the force-producing units of muscle, are built
(2000) and Riley et al. (2000) also paved the way for MRI and and added in parallel, increasing muscle cross sectional area”
delicate microscopic inquiry of muscle adaptation to training. (p. 207). Interestingly, Wisdom and colleagues cited work from
The aforementioned literature demonstrates that a common Johnson and Klueber (1991) and Farup et al. (2012) as evidence to
goal of several laboratories for over a century has been to support their hypothesis, although neither study directly assessed
assess how training affects indices representative of skeletal sarcomere abundance or myofibrillar protein concentration.
muscle hypertrophy. Yet, a clear definition of hypertrophy Upon activation, muscle contractile force is produced by myosin
is difficult to elucidate and depends on which literature, or and actin cross-bridge formation, the myosin power stroke,
scientist, is consulted. and transmission of tensile forces across the fiber (Tyska and
Warshaw, 2002; Karatzaferi et al., 2004). Therefore, at the single
fiber level, hypertrophy has been posited to coincide with the
WHAT ARE THE MOLECULAR maintenance of or increase in specific tension (N/cm2 ) through
UNDERPINNINGS OF SKELETAL the addition of sarcomeres in parallel (Wisdom et al., 2015;
MUSCLE HYPERTROPHY? Dankel et al., 2018). However, select evidence has failed to
confirm this relationship. For example, Meijer et al. (2015) have
In 2010, a co-author of the current review (B.J.S.) defined reported that lower specific tensions (N/cm2 ) exist in single fibers
skeletal muscle hypertrophy as an expansion of contractile isolated from bodybuilders with significantly larger fCSAs when
elements and extracellular matrix of skeletal muscle cells compared to muscle fibers from controls and power athletes.
(Schoenfeld, 2010), pointing to the addition of sarcomeres in Furthermore, in 1969 (although using a surgical ablation model
parallel and/or addition of myofibrils being largely responsible in rodents) Rowe reported ∼25% decreases in specific tension
for increases in cell size based on supporting evidence although CSAs of muscle samples significantly increased (Rowe,
from Paul and Rosenthal (2002) and Tesch and Larsson 1969). These conflicting observations could be explained by
(1982). Although this hypothesis is logical, neither investigation differential mechanisms of fCSA increases. For instance, fCSA
clearly provided counts of myofibrils or sarcomeres, which and cell mass can increase through an expansion of other
warrants reconsideration of the specific mode of short-term cell constituents in lieu of contractile protein changes (e.g.,
resistance training-induced hypertrophy in human fibers. Lloyd ICF, t-tubule or sarcoplasmic reticulum volume, sarcoplasmic
(2013) provided the elegantly simple definition of cell growth proteins, or mitochondrial volume). Indeed, as far back as 1976
as mass accumulation. Given the potential for conflicting Seiden reported robust increases in fiber diameters of rodent
definitions, it seems appropriate to operationally define skeletal muscle were largely associated with increases in sarcoplasmic
muscle hypertrophy as an increase in skeletal muscle mass. reticulum and t-tubule volumes in the context of “work-induced
However, this definition may oversimplify the compositional hypertrophy” while no apparent increases were observed in
alterations that occur during skeletal muscle hypertrophy in myofibril densities (Seiden, 1976). Also, it stands to reason that
response to resistance training that can affect muscle function. some individuals may realize significant increases or decreases
Alternatively stated, if hypertrophy is defined as an increase in myofibrillar protein content or myofibril number while
in mass, then this definition insufficiently communicates what others may not in response to the same training or unloading
specifically is altered within a skeletal muscle cell in response intervention. Research in humans in this regard seems equivocal
to resistance training and how the increase affects muscle (D’Antona et al., 2006; Trappe, 2009; Canepari et al., 2010;
function. Moreover, since the type of resistance training (e.g., Meijer et al., 2015).
chronic heavy versus light loading) likely results in specific A lack of evidence explicitly showing serial or parallel
compositional alterations, this consideration is vital to the sarcomere number increases in human fibers after resistance
comprehensive understanding of the specificity of skeletal training precludes the confident conclusion that myofibrillar
muscle adaptation. protein accretion is largely responsible for, or proportional to,
As stated above, resistance training-induced hypertrophy is increases in fCSA. In fact, upon extensive review of the literature,
thought to occur primarily through the addition of sarcomeres in it remains to be clearly determined if increases in sarcomere
series or in parallel in existent myofibrils (i.e., sarcomerogenesis), or myofibril number, and therefore protein abundance, strongly
or due to the synthesis of new myofibrils in existent muscle fibers correlate with increases in fCSA in response to short-term

resistance training in human fibers. Conversely, and as suggested Macroscopic Assessment

by MacDougall’s seminal 1982 publication, select evidence points Assessments of whole-body composition change are not the
to the contrary, at least in the short-term. For instance, Hody et al. focus of this review; however, certain whole-body and other
(2011)1 used proteomics to analyze VL muscle obtained from anthropometric assessments are commonly employed to infer
humans prior to and following 2 weeks of eccentric-emphasis that muscle hypertrophy has occurred. These whole-body
resistance training and reported a decrease in various contractile assessments will not be described or discussed in the same
proteins and an increase in the expression of oxidative enzymes. detail as more direct muscular assessments but deserve mention
As noted early on by Goldspink and Howells (1974), these and brief description in this section given their macroscopic
observations could be related to the specific dose of resistance nature and inclusion in Figure 3. These include air displacement
training. There are several reasons why this may be the case. For plethysmography (e.g., Bod Pod), hydrostatic weighing,
example, early adaptations to muscle fibers in response to novel bioelectrical impedance, skinfolds, and other anthropometrics
training stimuli may consist primarily of preparatory structural involving handheld measuring tapes and tools. These techniques
remodeling or metabolic changes that set the stage for subsequent are primarily used to estimate fat and fat-free mass. Since skeletal
growth. Also, cell damage experienced in response to a novel muscle tends to occupy a large percentage of fat-free mass,
training stimulus is associated with increased swelling/edema and an observed increase in fat-free mass based on these methods
this could affect assessments of protein abundance at various is commonly thought to indicate hypertrophy has occurred,
time points. Nevertheless, decades of anecdotal observations in although this is not necessarily the case given that many other
the practical setting of individuals realizing significant increases factors contribute to fat-free mass (e.g., fluid).
in muscle mass in response to chronic resistance training Air displacement plethysmography uses whole-body
suggests eventual increases in contractile protein abundance. densitometry to estimate body composition (fat and fat-free
Moreover, recent evidence from van der Pijl et al. (2018) in mice mass). Hydrostatic or underwater weighing measures mass
suggests increased sarcomeres in series and parallel resulting in per unit of body volume in order to calculate body density
hypertrophy in response to unilateral diaphragm denervation. and is based on Archimedes’ principle. Once body density is
Additionally, a host of studies employing tracer methods calculated, fat and fat free mass can be estimated. Bioelectrical
reporting significant increases in myofibrillar protein synthesis impedance estimates total body water by calculating opposition
rates in response to resistance training in the fasted and to the flow of an electric current. Fat and fat-free mass can be
fed states provide robust support for the notion that chronic estimated from total body water. Skinfolds are performed by
resistance training results in myofibrillar protein accretion pinching specific locations of skin and underlying subcutaneous
(Louis et al., 2003; Kim et al., 2005; Moore et al., 2005; adipose tissue relative to precisely measured anatomical
Cuthbertson et al., 2006; Carrithers et al., 2007; Kumar locations. Skinfold thicknesses can be summed and used to
et al., 2009, 2012; West et al., 2009; Burd et al., 2010; estimate body composition using various formulae. Technically,
Holm et al., 2010; Camera et al., 2012; Witard et al., anthropometrics encompasses any assessment of body size,
2014). Notwithstanding, while these tracer studies have been shape, and composition. However, in Figure 3, we are referring
insightful, it is noteworthy that the measurement of myofibrillar to the systematic measurement of segment girths in order
protein synthesis rates are not synonymous to measurements ascertain changes in size potentially due to hypertrophy. While
of myofibrillar protein concentrations, myofibril number, or each of these assessments can be considered macroscopic, they
sarcomere number. Moreover, it seems the investigation of do not directly assess muscle tissue.
ultrastructural and biochemical alterations to resistance training The focus of this section is on common measurements in
have been deprioritized in lieu of delicate tracer work. research studies which aim to directly assess muscle tissue and
Consequently, the hypothetical model of proportional increases intuit that muscle hypertrophy has occurred. A description of
in myofibrillar protein concentration concomitant with increases each measurement approach is outlined below in order to provide
in fCSA lacks robust evidence. With this in mind, macroscopic context as to how each measure differs and what the implications
and microscopic assessments of hypertrophy are briefly described of those differences may be. A host of measurement techniques
below while a comparatively greater breadth of attention is exist to assess hypertrophy in accordance with a set dimension;
devoted to the molecular assessment of hypertrophy in the that is, thickness (1D), CSA (2D), volume (3D), and mass.
“Molecular assessment” section that follows.
Muscle Thickness Assessment Using Ultrasound
Muscle thickness, as assessed by B-mode ultrasonography, is a
MEASUREMENT OF HYPERTROPHY rapid, easy, relatively inexpensive, and non-invasive assessment
of gross muscle size. To measure muscle thickness, investigators
For parsimony, we divide measurement techniques pertaining often image the mid-belly of a muscle and measure the linear
to each of these levels into the following categories: (a) distance between the deep and superficial aponeurosis of the
macroscopic measurements, (b) microscopic measurements, and muscle of interest (Franchi et al., 2018b). While muscle thickness
(c) molecular measurements. has been shown to be highly reliable in a range of muscles
(intra-class correlations, or ICCs = 0.65–0.94) (Thoirs and
1
5 sessions per week for 2 weeks consisting of 3 sets of 30 maximal voluntary English, 2009), it is limited in that it is only representative
contractions of the quadriceps muscle. of one dimension of the muscle. For example, relative to

FIGURE 3 | Different assessments used to monitor resistance training-induced adaptations. The diagramed techniques are utilized to measure whole-body
adaptations down to molecular adaptations to resistance training. Particular attention in this review is devoted to localized, microscopic, ultramiscroscopic, and
molecular assessments. Images are either from our laboratory or were obtained online where reuse for educational purposes was not restricted.
muscle thickness, muscle width, and length may hypertrophy regard to assessing fat-free/bone-free lean tissue mass during
differently, and proximal changes may be different than distal whole-body scans (e.g., >0.99) (Kephart et al., 2016). Unlike
changes. This point was recently demonstrated by Vigotsky et al. one-dimensional ultrasound assessments and other methods
(2018), who reported that hypertrophy of different regions of the discussed below, however, DXA has the inability to discriminate
same muscle are not strongly correlated within an individual. between muscle groups. Additionally, while DXA can distinguish
Additionally, ultrasound is highly dependent on the skill of the bone, fat, and fat-free/bone-free lean tissue, it cannot distinguish
investigator, given that differences in the pressure exerted by the muscle tissue from intramuscular fluid, nor is it sensitive enough
transducer against the skin can result in substantial variations to detect intramuscular fat. DXA can also be highly influenced
in measurements and thus high inter-rater error rates. Thus, by hydration status and other factors described by Nana et al.
ultrasound-based assessments of muscle thickness provide a fast (2015) in greater detail which also persuade careful methods
and practical assessment of 1D muscle size, but the quality of and interpretation. Notwithstanding, DXA can be used as a
these assessments may very well be rater-dependent. non-invasive assessment of gross and segmental lean body mass.
DXA CT
Dual-energy x-ray absorptiometry was originally designed to Computed tomography was introduced in the early 1970s
measure bone mineral parameters and is now a widely used (Hounsfield, 1973) and has the ability to provide high-contrast,
method to assess skeletal muscle mass changes. Whole-body 2D images with pixel intensities related to tissue density.
DXA scans render 2D images, although these scans can provide Tissues often measured include adipose and skeletal muscle
mass and density estimates when calibrated against a phantom (Heymsfield et al., 2014). When used as a measurement of muscle
of known density. Additionally, region of interest tools can be hypertrophy, it is common for images to be manually segmented
used to quantify appendicular lean masses. Newer DXA scanners for specific muscles or muscle groups and then quantified. CT
have been shown to produce excellent test-retest ICCs with is considered a reliable and valid method of assessing changes

in muscle CSA. For example, Verdijk et al. (2009) reported fields are used to localize those particles. Therefore, MRI is
coefficients of variation of 0.6% for repeated scans of muscle particularly useful for studying hydrogen-dense soft tissue, such
CSA using CT methods and inter- and intrarater reliability as adipose tissue and skeletal muscle. For MRI-based volume
coefficients of 0.996 and 0.997, respectively. A drawback to assessments, a series of 2D cross-sectional slices are obtained
CT scanning is that subjects are exposed to larger doses of and integrated as a function of distance to obtain volume (i.e.,
R ins
radiation relative to DXA along with being costly per scan V = orig ACSA(x)dx). Test-retest values for select upper and
(Prado and Heymsfield, 2014). lower-body muscles have yielded exceptionally high ICCs (e.g.,
0.99) (LeBlanc et al., 2000; Smeulders et al., 2010). Although
pQCT these measures are the most accurate in terms of capturing
Peripheral quantitative computed tomography (pQCT) was changes in gross muscle size, MRI equipment is not widely
originally developed for bone density (Gasser, 1995), but has accessible and the scans are costly; therefore, its use is scarcer
been validated to measure training-induced changes in muscle in the literature. Furthermore, while MRI is considered the gold
size (DeFreitas et al., 2010) and has been reported to largely standard for assessing the 2D area or segmental volume of a
agree with MRI measurements (R2 = 0.98). Beyond the work particular muscle group, it does not glean molecular adaptations
of DeFrietas et al. to our knowledge, there are no other studies that occur within fibers [e.g., changes in contractile protein
directly comparing pQCT with other measures in response to concentration, sarcoplasmic protein concentration, intra- versus
a hypertrophy stimulus pointing to the need for researchers to extracellular fluid (ECF), etc.] nor does it uncover the metabolic
consider this method of assessment in future work. A strength and functional nature of the tissue compared to other methods
of this measurement is the ability to detect intramuscular (Hellerstein and Evans, 2017).
fat concentration which could indicate skeletal muscle quality
(Sherk et al., 2014). Similar to DXA and CT, however, a Three-Dimensional Ultrasound (3DUS)
limitation of the pQCT is that it cannot distinguish between Three-dimensional ultrasound is a newer, promising approach
muscle tissue and intramuscular fluid. Therefore, it likely reflects to capturing 3D muscle architecture with standard B-mode
changes in contractile protein as well as potential alterations ultrasound. By combining ultrasonography with 3D motion
in training-induced fluid shifts or glycogen changes. There also capture, the location and orientation of each frame of an
seem to be no standardized protocols for imaging or analysis, so ultrasound video can be transformed into the lab coordinate
comparing studies is difficult (Erlandson et al., 2016). system (Mozaffari and Lee, 2017). Thus, the muscle boundaries
in each digitized frame can be reconstructed in 3D, from which
Panoramic/EFOV Ultrasound muscle volume (or area) can be calculated. These approaches
Panoramic and extended-field-of-view (EFOV) ultrasound are have been validated against MRI (ICC > 0.99) (Barber et al.,
new techniques that use traditional ultrasound B-mode imaging, 2009). In addition, 3DUS allows for the quantification of
but “stitch” together a series of images, so as to reconstruct fascicle-level geometry in 3D (Rana et al., 2013), which classically
a larger, wider 2D image. These techniques have primarily has been difficult to measure. Such insights show promise for
been applied for the assessment of anatomical CSA (ACSA) improving our understanding of not only muscle geometry, but
and fascicle length. Research shows that panoramic ultrasound also function (Franchi et al., 2018b).
displays a range of concordance correlation coefficients (CCC)
when estimating hypertrophy and atrophy compared to MRI Microscopic Assessment
(CCC = 0.37–0.78) (Scott et al., 2017). Axially, EFOV ultrasound Skeletal muscle hypertrophy has been assessed on the
has been shown to be both valid and reliable for assessing microstructural level using fCSA measurements and prepared
extensor carpi ulnaris fascicle lengths (Adkins et al., 2017). using histochemical staining after samples are sliced and attached
Additionally, mid-thigh area muscle assessments using EFOV to microscope slides. This technique has been used to evaluate
ultrasound and CT scans have been shown to agree well with each the structure and size of muscle samples since the late 1800s.
other (Noorkoiv et al., 2010). However, Bland-Altman plots have Gunnar Nÿstrom was one of the first to utilize these methods
demonstrated that panoramic ultrasound images from several in the evaluation of muscle tissue in mice by staining cardiac
muscle groups typically yield lower ACSA values compared to musculature with black India ink and using light microscopy
MRI values (Scott et al., 2012). These findings highlight the to examine the structure of the samples. While his interests
nuances required when interpreting results using whole muscle ultimately were the transverse tubules, he also found light
imaging, in that there are likely muscle- and dimension-specific and dark bands (isotropic and anisotropic bands, respectively)
differences in validity and reliability. stretching the distance of each sarcomere (Nystrom, 1897).
Shortly thereafter, the first study on skeletal muscle hypertrophy
MRI was conducted by Morpurgo examining the effect of run
Magnetic resonance imaging is non-invasive, has excellent training on skeletal muscle hypertrophy in dogs which had
resolution, allows discrimination between separate muscles, their left sartorius muscle removed before undergoing 2 months
and is commonly thought of as the reference standard for of training (Morpurgo, 1897). After sacrificing the dogs, the
regional muscle mass assessment (Smeulders et al., 2010). MRI right sartorius muscle was evaluated using light microscopy to
uses radio pulse waves to induce the nuclear spin of atomic examine sections adhered to slides with particular interest in
particles – particularly those in hydrogen – and electromagnetic fCSA change. Much of the work evaluating hypertrophy at the

cellular level between the late-1800s and the mid-1900s utilized potential to present an increase in fiber quantity and perhaps a
animal models, and similar to present day, the sectioning of decrease in mean fCSA when analyzing via microscopy.
muscle tissue for observation using microscopy. As mentioned Interestingly, fCSA adaptations to resistance training
previously, it was not until the Bergstrom needle was introduced are usually relatively greater than any other hypertrophy
in 1962 that these methods were utilized more openly in research surrogates when expressed as percent change (described in
related to skeletal muscle hypertrophy in humans. The Bergstrom the “Measurement Agreement” section). Regarding fCSA
method can yield tissue weights ranging from ∼25 to ∼300 mg. hypertrophy, consideration of the fiber type (e.g., I, IIa, and IIx)
Studies conducted prior to 1960 such as the aforementioned, seems warranted, as evidence has revealed fibers exhibit different
and those of the modern era share many of the same magnitudes of hypertrophy following periods of resistance
fundamentals of sample extraction and preparation with many of training (Fry, 2004). Reporting of mean fCSA, which accounts
the differences coming via technological advancement of imaging for all fibers, may result in different change scores compared to
programs and the introduction of computer based models to reporting type I or type IIa muscle fibers independently. Kosek
aid in evaluation. The procurement of muscle biopsies are safe, et al. (2006) reported that mean type II fCSA (which weights
minimally invasive, and can be performed as an outpatient the percent distribution of both IIa and IIx fibers) exhibited
procedure using the modified Bergstrom technique (Shanely ∼32% hypertrophy in young adult fibers following 16 weeks
et al., 2014). The basic methods of tissue processing are as follows of resistance training, yet type IIa fibers demonstrated ∼25%
(Verdijk et al., 2009; Mobley et al., 2017): (a) approximately hypertrophy and type I fibers ∼18%.
20–40 mg of tissue is typically placed into a cryomold with media Although a sensitive assessment of skeletal muscle
before slow freezing in isopentane cooled by liquid nitrogen and hypertrophy, there are also limitations of fCSA calculations,
subsequent storage at −80◦ C, (b) samples are cut in thin slices such as the method of tissue processing, biopsy location, and
ranging ∼5–20 µm thick and are adhered to a slide, and (c) measurement methods. For example, it is practically impossible
different methods can be used to stain the samples for evaluation to biopsy the same location in a muscle twice, so any changes
of mean fCSA or fiber type-specific fCSA such as those described in size observed are assumed to extrapolate to surrounding
in Haun et al. (2017), Roberts B.M. et al. (2018), Wang et al. fibers, or the same fibers along their length. The climate of a
(2015), and Verdijk et al. (2009). In general, light is emitted from laboratory, buffers, and other factors during tissue processing can
the microscope and passed through a filter to isolate a specific affect measurements of fiber size. For instance, since a muscle
frequency which is absorbed by the specimen, and nanoseconds cell is ∼70% fluid, tissue processing can create variability of
later, light is returned from the specimen (Stokes shift) which is water retention in the muscle sample; and this could potentially
filtered by selected band pass filters (FITC, DAPI, TRITC, etc.) to alter findings assuming unstandardized processing procedures
illuminate the image returned in a certain manner based on the from one sample or from one laboratory to the next. Muscle
fluorophores used in the staining process (Sanderson et al., 2014). glycogen concentration could also affect muscle fCSA, and this
Typically, the returned images are captured between 10 and variable often remains unreported in manuscripts providing
20× magnifications before being evaluated through specialized fCSA measurements. Additionally, muscles can hypertrophy in a
software or manual measurement, although different laboratories non-uniform manner (distal versus proximal), which would not
employ distinct techniques which have contributed to some of the be detected with single-site measurements (Narici et al., 1996).
variation in findings (further discussed below). Furthermore, a variety of fiber-sizing software and laboratory
The hypertrophic response to resistance training correlates methods described in the published literature could differentially
strongly to the volume of training as discussed by Schoenfeld affect percent change calculations. It is common for authors to
(2010), suggesting a dose response relationship exists. In this devote only a few sentences to description of how fibers are sized
regard, Tesch and Larsson (1982) found that muscle tissue upon image capturing. For example, some laboratories randomly
from the medial deltoid and the VL obtained from high select 25–50 fibers for manual sizing while others size 100 or
level bodybuilders exhibit larger slow twitch fiber fCSA values more using specialized software (Lau et al., 2018; Wen et al.,
compared to recreationally trained individuals, although fibers 2018). Unfortunately, this often leaves absent specific calibration
exhibiting fast twitch properties yielded similar fCSA values procedures, disclosure of the training protocol used for the rater,
between cohorts. Additionally, Meijer et al. (2015) reported and the specific software or technique used to calculate fCSAs. In
that bodybuilders completing moderate to high volume training this regard, it has been reported that VL type II fCSA increases
display greater mean fCSAs compared to untrained individuals from ∼6,000 to ∼8,400 µm2 in college-aged men following
and strength/power athletes. However, it is also important to 12 weeks of resistance training (Snijders et al., 2016), whereas
note that while all fiber types were larger, there was a significant our laboratory as well as Reidy et al. have reported that VL type
difference between type I fCSA of bodybuilders as compared II fCSA increases from ∼5,100–5,500 to ∼6,000–6,500 µm2
to untrained individuals and strength/power athletes, yet no during this same time course in this same population (Mobley
meaningful difference between type I fCSA of the untrained et al., 2017; Reidy et al., 2017b). Another independent laboratory
group and the strength/power group. Additionally, Eriksson et al. has reported that VL type II fCSA increases from ∼6,200
(2006) conducted a study in high level powerlifters, some of to 7,500 µm2 in college-aged men following 16 weeks of
which self-reported use of anabolic steroids, and found that there resistance training (Bellamy et al., 2014). Considering these
may be potential for fiber splitting along the length of a muscle apparent discrepancies, it stands to reason that research subject
fiber with chronic high intensity loading which could have the characteristics like previous or current activity levels and

nutrition habits could be contributing factors; while differences of resistance training did not (on average) present increases in
in the training interventions and tissue processing methods myofibrillar or sarcoplasmic protein concentrations (Roberts
between laboratories could also explain the disagreement. M. D. et al., 2018). Although assessed through TEM, it is
We feel that both more appropriate and transparent method notable that Toth et al. (2012) and MacDougall et al. (1982)
descriptions and standardization between labs can help resolve have both reported decreases in myofibril density concomitantly
some of these discrepancies moving forward. occur with fCSA increases following 18 weeks and 6 months
of resistance training, respectively, although Luthi et al. (1986)
Molecular Assessment reported no change in myofibril density following 6 weeks of
The molecular signature that coincides with skeletal muscle training. Additionally, the aforementioned work by Penman in
hypertrophy in response to resistance training has largely been the 1960s suggest reductions in myosin density occur following
understudied, but can be inferred by analyzing changes in brief periods of resistance training (Penman, 1969). Although
protein sub-fractions within biopsied tissue through differential in animal muscle samples, other authors have also reported
centrifugation protocols followed by simple biochemical assays no change or reductions in myofibril density in response to
(e.g., Bradford or bicinchoninic acid assays), polyacrylamide short-term resistance training-induced hypertrophy (Goldspink
gel electrophoresis, immunoblotting for specific proteins and Howells, 1974; Seiden, 1976). Furthermore, the studies
of interest, or larger-scale proteomic-based assessments. described previously showing lower specific tension (i.e., N/µm2 )
Although theoretically simple, these methods produce practical in larger fibers or hypertrophied fibers in response to resistance
challenges that often prevent laboratories from employing training seem to support a potential dilution of myofibrils.
them. However, we feel this area of inquiry is vital to our A summary of these human studies are presented in Table 1.
comprehensive understanding of skeletal muscle adaptation In interpreting these data, increases in myofiber size with
to training. The measurement of skeletal muscle myofibrils no change in myofibrillar protein concentration would indicate
dates back at least as far as 1957 when Hanson and Huxley that cellular growth occurs with proportional increases in
(1957) used extraction, solubilization, and polyacrylamide-gel myofibril protein accretion. That is, the stoichiometry of the
based separation techniques to quantify myosin and actin muscle cell would be mostly preserved in this case. Conversely,
concentrations. More recent studies have used various methods increases in myofiber size with a decrement in myofibrillar
to determine how resistance training affects myofibrillar and/or protein concentration would indicate that the protein pool is
sarcoplasmic protein concentrations, and given that assessment being diluted through increases in ICF or other sarcoplasmic
methods have been inconsistent, discordant findings have constituents. As mentioned above, select reports have curiously
resulted. For instance, Shelmadine et al. (2009) reported ∼50% suggested that resistance training robustly increases myofibrillar
increases in myofibrillar protein concentration following 28 days protein concentrations, which would indicate that substantial
of resistance training. Willoughby and Rosene (2001) similarly myofibrillar packing occurs within the first few weeks to
reported ∼40% increases in myofibrillar protein concentration months of training. Alternatively stated, such findings suggest
after 12 weeks of resistance training, and have also reported myofibril protein accretion far outpaces cell growth. Therefore,
∼85% increases in myofibrillar protein concentrations 6 h after these inconsistent reports on the molecular and ultrastructural
a single session of resistance training (Willoughby and Nelson, adaptations to resistance training warrant additional research.
2002). Cribb and Hayes (2006) and Cribb et al. (2007) have Beyond alterations in contractile and sarcoplasmic proteins,
reported similar increases in myofibrillar protein concentration potential fluid shifts are a commonly underappreciated
after 10 weeks of resistance training in two separate studies. molecular aspect of skeletal muscle adaptation to resistance
However, a comparatively greater number of authors have training. Sensitive assessment of skeletal muscle fiber fluid
reported no alteration in protein concentration or an apparent volume has traditionally been completed by weighing frozen,
decrease in response to resistance training. For instance, Brook hydrated samples on laboratory scales with ≤0.1 mg sensitivity,
et al. (2015) reported no significant change in total soluble freeze-drying samples in a vacuum-sealed benchtop apparatus,
protein concentration after 6 weeks of resistance training and calculating the difference between hydrated and dehydrated
(pre: 521 ± 34 mg/g, post: 552 ± 28 mg/g dry weight). Haus tissue weights after re-weighing on the same scale. Only a few
et al. (2007) reported no significant change in myofibrillar, investigations in humans have reported alterations in wet and
sarcoplasmic, myosin, or actin protein concentration after dry weights after a period of exercise training. For instance,
either 35 or 90 days of resistance training 2–3 days per week. Reidy et al. (2017a) reported significant increases in muscle
Woolstenhulme et al. (2006) reported no significant change tissue fluid content and fCSA after 12 weeks of resistance
in protein concentration after 8 weeks of resistance training training but no significant change in protein concentration.
although significant increases in type II fCSA occurred. Mora-Rodriguez et al. (2016) also reported significant increases
Carrithers et al. (2002) reported no significant change in in skeletal muscle sample water content but significant decreases
total, sarcoplasmic, myofibrillar, myosin, or actin protein in total protein concentration after 4 months of aerobic exercise
concentrations after 5 weeks of resistance training. Trappe et al. training. Harber et al. (2009) similarly reported significant
(2011) reported no significant change in the total percentage increases in muscle water content and significant decreases in
of water or protein content of biopsy samples after 12 weeks myofibrillar protein concentrations after 12 weeks of aerobic
of resistance training in older adults. Our laboratory recently exercise training, although fCSA measurements significantly
reported that high and low hypertrophic responders to 12 weeks increased. Given the potential influence fluid volume may exert

TABLE 1 | Human studies observing ultrastructural changes from muscle biopsy specimens with resistance training.
Author/Year Subject and group description Methods Outcomes
Penman, 1969 College age males (a) Eight weeks intervention Myosin density: ↓ in all groups Actin and
(a) Isotonic leg extension training (n = 2) (b) VL biopsies obtained prior to and following the myosin filament diameters: ↑ in all groups
(b) Isometric leg extension training (n = 2) intervention
(c) Run training (n = 2) (c) TEM examination of myosin fiber density,
distance between myofilaments, myosin
filament diameter, actin filament diameter
Penman, 1970 College age males (a) Ten weeks intervention fCSA: ↔ Distance between myosin
(a) Leg RT and running (n = 3) (b) VL biopsies obtained prior to and following the filaments: ↓
intervention
(c) TEM examination of myosin fiber density,
distance between myofilaments, myosin
filament diameter, actin filament diameter
MacDougall Untrained males (UT) and well trained (a) Histological methods for biceps brachii fCSA Type I fCSA: UT-pre = UT-post = WT
et al., 1982 bodybuilders and powerlifters (WT). (b) TEM for MF, SARCO and MITO areas Type II fCSA: UT-pre < UT-post = WT
(a) UT (n = 5) MF area: UT-pre > UT-post > WT
(b) WT (n = 7) (WT average training SARCO area: UT-pre < UT-post < WT
age = 7 years) MITO area: UT-pre > UT-post = WT
Luthi et al., Untrained males (n = 5) (a) Six weeks intervention, 3 days/week VL CSA (CT): ↑ fCSA: ↔
1986 (b) CT scan for CL CSA MF area: ↔
(c) Histology for VL fCSA MITO area: ↔
(d) TEM for MF and MITO areas
Willoughby and Untrained males (a) Twelve weeks intervention. Whole body RT MF protein concentration: ↑ in both groups
Rosene, 2001 (a) Supplemental creatine (n = 8) 3 days/week
(b) Placebo (n = 8) (b) Biochemical assays used for VL MF protein
which was isolated using TRIzol-based
methods (no histology)
Carrithers et al., Untrained males and females (a) VL and soleus biopsies completed prior to and VL total protein: ↔ in any group
2002 (a) Unilateral Limb Suspension (ULLS) following 5-week intervention VL cytosolic protein: ↔ in any group
(n = 11) (b) Biochemical assays for either total protein, VL MF protein: ↔ in any group
(b) Resistance training (RT) (n = 10) cytosolic protein, myofibrillar protein, myosin VL myosin concentration: ↔ in any group
(c) ULLS+RT (n = 10) concentration, and actin concentration VL actin concentration: ↔ in any group
Other notes: total protein, cytosolic protein
and MF protein decreases were observed
in the soleus muscle of the ULLS group
Cribb and Trained males (a) Ten weeks whole body RT, 3 days/week fCSA (type I and II): ↑ in both groups
Hayes, 2006 (a) PRE/POST (n = 8) (b) VL biopsies prior to and following the 10-week MF concentration: ↑ in both groups
(b) MORN/EVE (n = 9) intervention
(c) Histological methods for VL fCSA
(d) Biochemical assays used for MF protein
Cribb et al., Trained males (n = 31) (a) Ten weeks whole body RT, 3 days/week fCSA (type I and II): ↑ in all groups,
2007 (a) PRO (n = 10) (b) VL biopsies prior to and following the 10-week Cr-PRO-CHO greater type II increase after
(b) PRO-CHO (n = 11) intervention 10 weeks
(c) Cr-PRO-CHO (n = 10) (c) Histological methods for VL fCSA MF concentration: ↑ in all groups,
(d) Biochemical assays used for MF protein Cr-PRO-CHO greater increase after
10 weeks
Woolstenhulme Untrained males (a) Untrained males performed either 8 weeks Type I fCSA: no difference between trained
et al., 2006 (a) Single bout (n = 6) lower body RT 3 days/week or single bout of and single-bout
(b) Eight weeks RT (n = 6) RT Type II fCSA: ↑ in trained, but ↔ in single
(b) VL biopsies prior to and following 8-week bout
intervention Total muscle protein concentration: ↔ in
(c) Biochemical methods for total protein either group desmin protein concentration:
concentration ↑ in trained, but ↔ in single bout actin
(d) Histology for fCSA protein concentration: ↔ in either group
(e) Immunoblotting for desmin, actin, and Dystrophin protein concentration: ↔ in
dystrophin either group
(Continued)

TABLE 1 | Continued
Haus et al., 2007 Untrained males (a) Subjects completed either 35 days ULLS, 35 days ULLS: ↓ in muscle volume
(a) Unilateral Limb Suspension (ULLS) 35 days ULLS+RT, 90 days BR, 90 days 35 days ULLS+RT: ↑ in muscle volume
(n = 11) BR+RT. 90 days BR: ↓ in muscle volume
(b) ULLS+RT (n = 10) (b) Assessments prior to and following either 35 or 90 days BR+RT: ↔ in muscle volume
(c) Bed rest (BR) (n = 9) 90 days which included VL muscle biopsies All groups: ↔ mixed protein, SARCO
(d) BR+RT (n = 8) and mid-thigh MRI protein, MF protein, myosin, actin, or
(c) Biochemical methods for assessment of collagen protein concentrations
muscle protein quantification as mixed,
sarcoplasmic, and myofibrillar
(d) SDS–PAGE methods for assessment of
myosin, actin, and collagen protein
concentrations
Shelmadine et al., Untrained males (a) Four weeks intervention, 2 days/week upper MF concentration: ↑ in both groups
2009 (a) Supplemental pre-workout (n = 9) and 2 days/week lower split
(b) Supplemental placebo (n = 9) (b) Biochemical assays used for VL MF protein
which was isolated using TRIzol-based
methods (no histology)
Trappe et al., 2011 Older adults (n = 36) (a) Twelve weeks knee extensor resistance Quadricep muscle volume: ↑ in all
(a) placebo (n = 12) exercise groups, acetaminophen and ibuprofen
(b) acetaminophen (n = 11) (b) MRI measurement of quadricep muscle volume ↑ more than placebo
(c) ibuprofen (n = 13) (c) VL biopsy prior to and following 12 weeks Muscle protein content: ↔ in all groups
intervention Muscle water content: ↔ in all groups
(d) Biochemical assays used for muscle protein
and water content (% muscle wet weight)
Toth et al., 2012 (a) Heart failure patients (HFP) (n = 10) (a) Eighteen weeks whole body RT fCSA (type I and II): ↔ in both groups
(b) Minimally active people (CTL) (b) VL biopsies prior to and following 18-week MF area: ↓ in both groups
(n = 14) intervention A-band length: ↑ in both groups
(c) Single muscle fiber morphology
(cross-sectional area)
(d) Electron-microscopy-based ultrastructural
measurements
(e) Single fiber mechanical measurements.
Brook et al., 2015 Untrained males (n = 10) (a) Assessments prior to and following 6 weeks of Thigh lean mass: ↑ in T but not UT
unilateral leg RT (noted as T), with contralateral VL thickness: ↑ in T but not UT
leg serving as control (noted as UT) VL fiber length and pennation angle: ↑
(b) VL biopsies prior to, middle, and following the in T but not UT
intervention Myofibrillar FSR: ↑ in T but not UT
(c) Mid-thigh muscle architecture and DXA-derived VL total protein: no difference between
mass T and UT
(d) VL myofibrillar fractional synthesis rate
(e) VL total protein, DNA, RNA concentrations
using spectrophotometry
Reidy et al., 2017a Untrained males (n = 31) (a) Twelve weeks whole body RT fCSA: ↑
(b) VL biopsies prior to and following 12-week Post-absorptive MPS: ↑
intervention Post-absorptive MPB: ↓
(c) Histological methods for VL fCSA Muscle protein concentration: ↔
(d) Post-absorptive MPS and MPB assessments Muscle water content: ↑
(e) Biochemical assays for muscle protein VL thickness: ↑
concentration, DNA concentration, water Leg volume: ↑
content Leg lean mass: ↑
(f) VL Ultrasound, MRI, and DXA
Roberts M. D. Untrained males (a) Twelve weeks whole body RT fCSA (type I and II): ↑ in HI, ↔ in LO
et al., 2018 (a) High hypertrophic responders (HI) (b) VL biopsies prior to and following 12-week MF concentration: ↔ in HI, ↔ in LO
(n = 13) intervention Myosin and actin concentration: ↔ in
(b) Low hypertrophic responders (LO) (c) Histological methods for VL fCSA HI, ↔ in LO
(n = 12)
(Continued)

TABLE 1 | Continued
Assessed using combined metrics. (d) Biochemical assays used for MF protein, SARCO concentration: ↔ in HI, ↔ in
SARCO protein and MITO volume LO
(e) SDS–PAGE for actin and myosin content MITO content: ↔ in HI, ↔ in LO
Unpublished Previously trained college age males; (a) Six weeks whole body high volume training fCSA (type I and II): ↑
data from Haun only high hypertrophic responders (HI) 3 days/weeks Myosin and actin concentration: ↓
et al., 2018 represented (n = 15) (b) VL biopsies prior to, middle, and following the Phalloidin staining intensity/fiber: ↓
Response determined VL mean fCSA intervention SARCO protein concentration: ↑
increases (c) Histological methods for VL fCSA (p = 0.065)
(d) Biochemical assays used for SARCO protein MITO content: ↓
and MITO volume
(e) SDS–PAGE for actin and myosin content
(f) Phalloidin staining for contractile protein
content per fiber
These studies used transmission electron microscopy (TEM) or biochemical techniques to assess changes in myofibrillar protein content in muscle biopsy specimens
following weeks to months of resistance training (RT). Bold-faced text indicates significant effects were noted. Symbols and other abbreviations: 1, pre-study versus
post-study value; ↑, significant increase; ↓, significant decrease; ↔, no change from a statistical standpoint; MF, myofibrillar fraction; SARCO, sarcoplasmic fraction; VL,
vastus lateralis; fCSA, fiber cross-sectional area; SDS–PAGE, polyacrylamide gel electrophoresis.
on fCSA measurements used to surmise hypertrophy after an There are also differences when comparing the degree
exercise intervention, it seems critical that water content be of hypertrophy between MRI and biopsy fCSA metrics. For
accounted for when analyzing microscopic and molecular-level example, Narici et al. (1996) reported a 2% increase in VL
hypertrophy. However, it should be noted that the freeze-drying fCSA, but a 7% increase in VL CSA measured by MRI
method cannot delineate ICF from ECF levels. Thus, other following 6 months of resistance training. These authors
techniques are needed to determine these metrics, and this is reported that differences existed when comparing either of those
discussed in greater detail below. two measurements to whole quadriceps measurements, which
increased by 15%. Another study by Aagaard et al. (2001)
compared changes in VL muscle size using MRI, biopsy, and
MEASUREMENT AGREEMENT ultrasound after 14 weeks of resistance training. These authors
reported a ∼16% increase in fCSA, yet only a ∼10% increase
While the direct comparison of different methods of in muscle volume. Additionally, a positive relationship existed
hypertrophic assessment are sparse, there are data suggesting between the change in fCSA and CSA assessed via MRI, although
that macroscopic, microscopic, ultramicroscopic and/or this correlation was only moderate (r = 0.58). As previously
biochemical indices of skeletal muscle hypertrophy following mentioned, several studies have also noted fCSA increases in
resistance training have poor agreement. A recent study that response to resistance training yield greater relative change
perhaps best demonstrates this phenomenon is by Franchi et al. scores compared to other hypertrophy surrogates. For example,
(2018a), who observed that, while percent change increases Esmarck et al. (2001) reported a 7% increase in VL CSA assessed
in ultrasound-assessed VL muscle thickness and MRI-derived by MRI and a 22% increase in VL fCSA following 12 weeks of
CSA showed strong correlations following 12 weeks of leg resistance training. Verdijk et al. (2009) reported an 8.5% increase
extensor resistance training (r = 0.69), ultrasound findings were in quadriceps CSA assessed by CT, but a 28% increase in Type II
poorly associated with MRI-derived calculations of muscle fCSA. Frontera et al. (1988) reported a 10% increase in quadriceps
volume (r = 0.33). DXA is highly correlated with both MRI and CSA assessed by CT, but ∼28% increase in VL fCSA. Moreover,
CT measures of muscle area when assessed at a single point when analyzing data from a recently published study from our
in time (Levine et al., 2000; Maden-Wilkinson et al., 2013). laboratory (Haun et al., 2018), the top 10 hypertrophic responders
However, DXA shows only a moderate correlation with CT to 6 weeks of high-volume resistance training experienced a 23%
(r = 0.52) when assessing changes in muscle mass following increase in right VL fCSA, although upper right lower extremity
regimented resistance training, and its high measurement error lean mass assessed by DXA only increased by 8.8% and mid-thigh
raises questions as to suitability for determining subtle changes thickness assessed via ultrasound only increased by 2.1%.
in muscle mass over the course of a regimented resistance When associating ultrastructural and histological adaptations
training protocol (Delmonico et al., 2008). Consistent with to resistance training, there is no clear relationship. As stated
this hypothesis, Snijders et al. (2015) reported statistically above, the landmark TEM work by Penman and MacDougall
greater increases in MRI-assessed thigh muscle CSA in a suggests that the concomitant dilution of myofibrillar proteins
protein-supplemented versus placebo group following a 12-week with fCSA increases may occur with resistance training.
resistance training program; however, lower extremity lean Remarkably, these data agree with studies performed decades
mass as measured by DXA failed to show statistically significant later. For instance, Toth et al. (2012) reported that 18 weeks
changes from pre- to post-study. of resistance training resulted in a statistically significant

13% decrease in VL myofibrillar protein area (assessed via body strength [assessed via three repetition maximum (3RM)
TEM), although no changes were noted in fCSA. Additionally, back squat] relative to LO responders (mean ± SE: 13RM
Harber’s group reported 12 weeks of cycle ergometer training squat = +42 ± 3 kg, LO 13RM squat = +31 ± 9 kg; p = 0.005),
in older, untrained subjects increased fCSA by ∼20%, increased and we noted an advantage of our clustering method included
quadriceps CSA (assessed via MRS) by 12%, and increased its association with a functional strength outcome. On average,
knee extensor power by 55% (Harber et al., 2009). Yet, pre-to-post training changes in VL fCSA were +1426 ± 253 µm2
myofibril protein concentrations (assessed through biochemical (mean ± SE) in HI responders and +5 ± 209 µm2 in LO
methods) decreased by ∼14%. Yet, Harber’s group has also responders (p < 0.001). However, no significant between- or
shown that elite runners myofibrillar protein concentrations within-cluster changes in myofibrillar protein concentrations
are higher than recreational runners (Reidy et al., 2014). were observed, and regardless of response cluster, there was
Observations of increased micro-/macro-structural variables not an association between pre-to-post training changes in
indicative of skeletal muscle growth along with improvements fCSA and myofibrillar protein concentrations (r = −0.014,
in functional performance metrics led the authors to justifiably p = 0.947). Table 2 displays Pearson’s r correlation values between
conclude aerobic exercise training resulted in significant muscle whole-body, regional, microscopic, and ultrastructural indices of
hypertrophy. Nevertheless, it is intriguing that this study similarly skeletal muscle hypertrophy from the subjects of this study.
observed various indices of muscle hypertrophy occurred in lieu Notably, the data in Table 2 includes percent change scores
of decrements in myofibrillar protein levels. in DXA TBMM, VL thickness, VL fCSA, VL myofibrillar
Importantly, many of the supposedly discrepant findings protein concentrations, VL sarcoplasmic protein concentrations,
compared above originate from different methods and this will be and VL myosin protein concentrations. It is apparent that
further discussed in a later section. However, the aforementioned ultrastructural indices of skeletal muscle hypertrophy typically
studies in this section demonstrating that different surrogates agree well with one another. For instance, moderate correlations
of hypertrophy seemingly disagree with each other prompted exist for raw delta as well as percent change scores in myosin
our laboratory to examine VL myofibrillar protein concentration protein and myofibrillar protein concentrations (r = 0.61).
differences between high versus low anabolic responders Moderate correlations also exist for raw delta as well as percent
following a 12-week full body resistance training program change scores in myosin protein and sarcoplasmic protein
(Roberts M. D. et al., 2018). Response clusters were generated concentrations (r = 0.71). However, microscopic assessments do
based on pre- to post-training changes in right VL muscle fCSA not agree well with ultrastructural assessments or macroscopic
(type I + type II fibers), VL thickness assessed via ultrasound, and assessments, and vice versa. To this point, we also performed
total body muscle mass (TBMM) assessed via DXA. Participants simple correlations for each of the surrogate measures of
in the upper and lower 25th percentiles were classified as high hypertrophy on data from all subjects regardless of cluster
(HI, n = 13) and low (LO, n = 12) hypertrophic responders, for greater statistical power (aside from myofibrillar and
respectively. Notably, this clustering method indicated HI sarcoplasmic protein concentration changes due to lack of
responders presented significantly greater increases in lower available sample from this study). Briefly, changes in VL thickness
TABLE 2 | Associations between macro-, micro-, and ultrastructural surrogates of hypertrophy following 12 weeks of resistance training.
1 DXA 1 VL 1 mean 1 MF 1 SARCO 1 myosin

TBMM (%) thick (%) fCSA (%) protein (%) protein (%) protein (%)
Mean values (SD) (n = 25) +5.9 (3.6) +17.5 +17.6 +3.6 (39.5) +6.8 (22.9) +10.5 (44.7)
(11.6) (23.5)
p-value relative to PRE <0.001 <0.001 0.002 0.660 0.284 0.685
correlation r-values
1 DXA TBMM (%) − 0.47 0.54 −0.08 −0.39 −0.20
1 VL thick (%) 0.47 − 0.31 0.37 −0.10 0.05
1 fCSA (%) 0.54 0.31 − 0.00 −0.04 0.21
1 MF protein (%) −0.08 0.37 0.00 − 0.45 0.61
1 SARCO protein (%) −0.39 −0.10 −0.04 0.45 − 0.71
1 myosin protein (%) −0.20 0.05 0.21 0.61 0.71 −
These data from our laboratory (Roberts M. D. et al., 2018) are pre- versus post-training percent change scores from 25 untrained, college-aged male participants that
engaged in 12 weeks of structured resistance training. Symbol and abbreviations: 1, pre- to post-training percent change; SD, standard deviation; DXA TBMM, dual x-ray
absorptiometry total body muscle mass; VL thick, ultrasound-assessed vastus lateralis thickness; fCSA, muscle fiber cross-sectional area; MF protein, myofibrillar protein
assessed through biochemical methods; SARCO protein, sarcoplasmic protein assessed through biochemical methods. Other notes: myosin protein was assessed
through electrophoretic methods with relative quantification being calculated through band densitometry. Findings: DXA TBMM, VL thick and mean fCSA metrics all
indicated that macro- and microscopic indices of skeletal muscle hypertrophy occurred. However, no strong correlations (i.e., r-values > 0.80) were observed between
percent change scores for these metrics. No significant changes in MF protein, SARCO protein or myosin protein occurred indicating that (on average) myofibrillar
packing or dilution did not occur. There were moderate correlations (r-values > 0.50) between 1 MF protein and 1 myosin protein as well as 1 myosin protein and 1
SARCO protein indicating that biochemical assessments generally were better associated with each other relative to macro- and microscopic assessments. All moderate
correlations are bold-faced.

produced correlation coefficients ranging from r = 0.06–0.25 in size including muscle thickness, muscle mass, and muscle
relation to changes in fCSA and DXA TBMM. Changes in type I volume. Yet, the current state of the evidence tells a different
and type II fCSA correlated weakly with changes in DXA TBMM and relatively inconsistent story. Based upon the current available
(r = 0.18, r = 0.13, respectively). evidence, Figure 4 summarizes how resistance training-induced
Considering these findings against the background of the skeletal muscle hypertrophy occurs at the macroscopic level and
current hypothetical model of RT-induced skeletal muscle may occur at the ultrastructural level.
hypertrophy presents a conundrum. For decades, it has been
assumed that the deposition of sarcomeres in parallel in existent
myofibrils, or the genesis of new myofibrils in existent muscle POTENTIAL EXPLANATIONS AND
fibers results in the observed expansion of fCSA and macroscopic LIMITATIONS
assessments of muscle size in response to RT interventions.
However, it is clear from the above data that this assumption The first and most obvious drawback when comparing biopsy
lacks consistent and explicit empirical support. After performing data to regional or whole body metrics is that the biopsy
an extensive search of the scientific literature, it is apparent specimen is small (e.g., 100 mg) relative to the VL muscle,
that no studies have directly quantified sarcomere number in and more so, relative to the musculature of the whole body.
parallel prior to or following resistance training in human In relation to characterizing ultrastructural indices using TEM,
fibers. Furthermore, the few studies employing TEM methods inadequate sampling is an imminent concern given that dozens
to provide myofibril densities were severely underpowered (not hundreds) of partial fiber areas are typically analyzed (Alway
having analyzed few fibers and few subjects, and although et al., 1988). Another viable concern with small samples from
critical first steps, do not allow confident population-wide both fCSA and TEM is a regression to the mean phenomenon
inferences. Strikingly, of the available evidence surveyed wherein from sampling sites, since it is impossible to biopsy the same
molecular and microscopic measurements occurred prior to location twice. This can make repeated measures difficult to
and following resistance training, a reduction in myofibrillar correlate because large or small values can skew results (Barnett
protein concentrations concomitant to observed increases in et al., 2005). Further compounding this issue is the lack of true
fCSA has been a more common finding. According to the widely controls in most training studies to help correct for repeated
assumed model of resistance training-induced hypertrophy, measures sampling complexity.
a maintenance of myofibrillar protein concentration should With regard to biochemical assays to determine myofibrillar,
coincide with an increase in fCSA. Moreover, this occurrence sarcoplasmic, myosin, and actin protein concentrations, we
should produce an increase in macroscopic measures of muscle recently reported that an adopted method from Alfred Goldberg’s
FIGURE 4 | Mechanisms of resistance training-induced skeletal muscle hypertrophy. Numerous studies have demonstrated that resistance training increases muscle
thickness (assessed using B-mode ultrasound) as well as muscle CSA (assessed with CT or MRI) (A). Likewise, numerous studies have reported that fCSA increases
occur with resistance training (B). However, the ultrastructural and molecular adaptations to resistance training remain largely unresolved (C).

laboratory (Cohen et al., 2009) yields good separation of Hellerstein and Evans (2017) deemed the ‘Virtual Biopsy’ shows
contractile and non-contractile proteins and is sensitive to detect promise in this regard by assessing whole-body muscle mass
5–25% changes in protein concentrations (Roberts M. D. et al., using metabolic labeling techniques for flux-rate measurements
2018). However, it is currently difficult to decipher how resistance in humans which focus on skeletal muscle, specifically.
training affects subcellular protein concentrations given that When interpreting and contrasting the results of different
multiple methods have been used (Willoughby et al., 2002; Cribb gross-level measurements, the dimensionality of each needs to be
and Hayes, 2006; Shelmadine et al., 2009; Roberts M. D. et al., considered. Of course, volumes, areas, and thickness are three-,
2018), and some of these reports using TRIzol-based methods two-, and one-dimensional, respectively, but understanding what
have likely not accurately reported myofibrillar protein levels this means in the context of hypertrophy requires deeper thought.
given that this method (Kopec et al., 2017): (a) leads to inefficient Starting from a unidimensional level, muscle can grow in
protein retrieval due to poor solubilization of precipitated protein three different, orthogonal directions. For instance, it may be
pellets, and (b) does not contain a sufficient detergent (e.g., possible that a muscle grows wider but not thicker, or has
Triton) to lyse membrane structures. In addition to these points, differential growth in different directions or parts of the muscle.
although mitochondrial volume has been consistently shown A unidimensional measure, such as thickness via ultrasound,
to decrease in response to resistance training (Groennebaek is inherently limited in capturing these possibilities; changes
and Vissing, 2017), alterations in sarcoplasmic reticulum and in off-axis lengths will not be captured. Area can, to some
t-tubule volumes have been largely unexplored in human skeletal extent, account for some of these differences, in that it captures
muscle in response to training interventions; and changes growth across an entire plane. If absolute growth does occur in
in these structures could contribute to observed increases or off-axis directions (e.g., a muscle not only grows thicker, but
decreases in fCSA. Moreover, disproportionate increases in ICF also wider), or even on parallel axes, we should not expect area
volume may occur with resistance training, albeit there is no to scale linearly with thickness. If thickness predominates, then
current microscopic or biochemical method to directly decipher a linear relationship between thickness and area should hold
this phenomenon. (Franchi et al., 2018a). Conversely, volume not only takes into
Limitations to various muscle imaging techniques also exist. account changes in muscle length, but also heterogeneities in
For instance, although MRI can estimate total fluid content area along the muscle. For instance, a muscle may grow more
within a scan (Ogino et al., 1994), standard MRI, x-ray, and distally than proximally. Such heterogeneities are best captured
ultrasound measurements cannot account for potential ECF and by taking advantage of the full dimensionality of the construct
ICF shifts that may occur with training. This is a critical point itself – muscle is three-dimensional. This may be why thickness
that is oftentimes underappreciated given that ECF, which does changes scale with area, but not volume (Franchi et al., 2018a).
not represent the intracellular milieu and contributes to mass Unfortunately, our understanding about the dimensionality and
and thickness changes, significantly increases with higher-volume heterogeneity of growth itself is limited; these properties may be
resistance training (Haun et al., 2018). Thus, the tandem use muscle and protocol-specific.
of regional bioelectrical impedance spectroscopy (BIS) with It is also noteworthy that muscle CSA assessment using
regional (e.g., thigh muscle area or volume) or whole body scans imaging techniques can be measured as one of two constructs
is a fruitful area for exploration. In this regard, we recently including ACSA and physiological CSA (PCSA). The former
used DXA to determine that 6 weeks of voluminous resistance can be assessed at any point along the muscle, and is often
training increased whole-body DXA LBM by +1.34 kg from defined to be the CSA orthogonal to the longitudinal axis of the
weeks 1 to 3 and an additional +0.85 kg from weeks 4 to 6 segment on which the muscle is located (i.e., in the transverse
in 30 previously trained college aged males (Haun et al., 2018). plane). Alternatively, PCSA is the muscle area orthogonal to a
When adjusting pre- to post-training changes in DXA LBM for muscle’s fibers; therefore, greater discrepancies will exist between
BIS-extrapolated ECF changes, however, a significant increase ACSA and PCSA with greater pennation. In contrast to ACSA,
occurred from weeks 1 to 3 (+1.18 kg), but not from weeks PCSA is assessed by measuring muscle volume and dividing it
4–6 (+0.25 kg). Hence, using methods that accurately account by average fiber length (usually at optimal length). Therefore,
for ICF and ECF shifts which occur during resistance training PCSA is a measure of the average CSA orthogonal to the
may provide more insight as to whether true hypertrophy fiber orientation and is seemingly related to the number of
occurs. Although BIS-based calculations of ECF and ICF have sarcomeres in parallel (Lieber and Ward, 2011). Although PCSA
been shown to strongly agree with sodium bromide dilution is more related to a muscle’s function than ACSA, the latter is
and deuterium oxide-based assessments (Moon et al., 2008; more commonly reported, primarily due to the methodological
Birzniece et al., 2015), it remains to be investigated how valid and limitations of obtaining PCSA.
reliable BIS-based assessments are compared to other methods
in context of resistance training-induced hypertrophy and fluid
alterations. Furthermore, Reidy et al. (2017a) have reported DEFINING SKELETAL MUSCLE
∼4 % increases in plasma volume after 12 weeks of resistance HYPERTROPHY AND FUTURE
training and a significant decrease in the percentage of muscle DIRECTIONS
water (−0.1–1.3%) which also persuades the measurement of
whole-body and muscle biopsy sample fluid content for more With intent to provide future research directions and improve the
accurate inference. A relatively new technique proposed by likelihood of fruitful discovery moving forward, it is necessary to

operationally define skeletal muscle hypertrophy in an objective rather than concluding “skeletal muscle hypertrophy” occurred
manner. To serve as rationale for our proposed definition and alone. Alternatively stated, we feel clear language pertaining to
types of skeletal muscle hypertrophy, we encourage readers the outcome data of a method should be explicitly reported to
to consider that various types of hypertrophy have been better portray the nature of a specific measurement. Second,
characterized in both cardiac and smooth muscle (Johansson, if multiple indices of skeletal muscle hypertrophy are being
1984; Mihl et al., 2008), although the construct of hypertrophy collected, then it would be valuable to include associations
is consistent in these definitions. between the measures in order to provide the reader greater
We propose that skeletal muscle hypertrophy be generally and insight as to how well or poorly the measurements agree.
simply defined as an increase in skeletal muscle size accompanied Third, when possible, we posit that using methods to determine
by an increase in mineral, protein, or substrate abundance (e.g., regional fluid shifts that account for ICF and ECF changes
glycogen and intramuscular triglyceride). However, considering could better delineate the mode of hypertrophy and whether
the reviewed evidence, we encourage the formal adoption of mass changes in a region of interest were largely due to fluid
three types of skeletal muscle hypertrophy worthy of further accumulation. Certainly, this assumes the research question
inquiry: (a) connective tissue hypertrophy, (b) myofibrillar centers around true protein accretion and not simply an
hypertrophy, and (c) sarcoplasmic hypertrophy. Connective expansion of other microscopic or macroscopic assessments as
tissue hypertrophy can be defined as an increase in the volume dependent variables, specifically.
of the extracellular matrix of skeletal muscle accompanied by We recently reported ECF-corrected LBM measures to better
an increase in mineral or protein abundance. Sarcoplasmic characterize hypertrophic responses to RT beyond ECF retention
hypertrophy can be defined as a chronic increase in the potentially due to edema. Although surface electrode BIS
volume of the sarcolemma and/or sarcoplasm accompanied possesses limitations given that greater subcutaneous adipose
by an increase in the volume of mitochondria, sarcoplasmic tissue thickness values can negatively influence skeletal muscle
reticulum, t-tubules, and/or sarcoplasmic enzyme or substrate impedance readings (Tagliabue et al., 2001), a newer fine
content. Myofibrillar hypertrophy can be defined as an increase needle BIS approach using small subcutaneous needles to bypass
in the size and/or number of myofibrils accompanied by an subcutaneous fat holds promise for more accurate ICF and ECF
increase in sarcomere number or sarcomeric protein abundance assessments (Kwon et al., 2017). Another viable strategy worthy
directly related to the structure or contractile force generation of consideration is to include multiple indices of skeletal muscle
of the sarcomere. hypertrophy at various levels. As an example, our laboratory has
Considering these definitions, we feel it is critical to appreciate implemented K-means cluster analysis based solely upon changes
the fact that measurement techniques assess different constructs in VL thickness to generate low, moderate, and high anabolic
and these differences do not necessarily make a measurement response clusters to 12 weeks of resistance training (Mobley et al.,
better or worse, but simply different. Resource constraints, 2018). As discussed earlier, we subsequently adopted a different
technical capabilities, and potential risks to participants are approach in these same subjects by generating clusters based
factors that inherently affect the selection of assessments of upon a composite hypertrophy score which entailed percent
muscle hypertrophy. To dismiss methodologies that provide changes in DXA TBMM (which only considers appendicular lean
less resolution of molecular changes based solely on this fact mass changes), VL thickness using ultrasound, and fCSA, and
is inappropriate as good reliability of a test can be incredibly selected high and low hypertrophic responders whose composite
useful for examining changes over time and inferring effects of scores existed in the upper and lower quartiles (Roberts M. D.
resistance training interventions. That is to say, while macro- and et al., 2018). Notably, in the former publication we did not
microscopic tests do not directly assess myofibrillar protein observe a between-cluster interaction for 3RM squat strength
accrual and fCSA, increases in these variables should result in although, as stated above, our latter publication yielded a
eventual increases in macroscopic indices. Stated differently, cluster × time interaction for 3RM squat strength whereby
while some methods of detecting true hypertrophy are more strength gains were greater in high responders. Likewise, a
vivid (e.g., myofibrillar protein), lower resolution methods similar multi-variable cluster approach has been adopted by
aren’t useless as they would almost certainly be corollaries of Davidsen et al. (2011), and more recently by Morton et al. (2018)
true hypertrophy. to stratify high-responders and low-responders to resistance
While it can be difficult to reconcile why different methods training. Thus, if clustering subjects in hopes of characterizing
used to assess skeletal muscle hypertrophy in response to specific phenotypes or predicting adaptive responses thereof is
resistance training do not always agree, we posit that certain the nature of the research question, we feel using a composite
procedures can be adopted to clarify research findings in hypertrophy score containing multiple indices (e.g., fCSA +
the field. First, if a single measure of hypertrophy is being DXA data + limb circumference + muscle thickness, etc.)
examined (e.g., VL thickness at the mid-thigh) and is found may be a viable approach. This approach can be thought of
to increase in response to training, then we view reporting as a form of dimensionality reduction; hypertrophic responses
that mid-thigh VL thickness increased in response to resistance can be measured in a number of ways, so procedures such as
training rather than stating muscle hypertrophy occurred is a principal component analysis can yield values that are linear
more accurate representation of the data. That is, unless multiple combinations of the predictor variables of interest, and a way that
levels of measurement are adopted along with assessment of fluid can account for much of the variance. However, as pointed out by
alterations, we encourage the reporting of the measurement itself Rucker et al. (2015), researchers should thoughtfully consider the

TABLE 3 | Test-retest reliability statistics for macroscopic, microscopic, and molecular assessments of hypertrophy.
Variable n CV of ICC Absolute SEM 95% CI Relative SEM (%) 95% CI (%)
measurement (%)
DXA LBM 10 0.95 1.00 0.47 kg 0.92 kg 0.92 1.81

VL thick 30 1.33 0.99 0.04 cm 0.08 cm 1.32 2.59
Mid-thigh circum. 10 0.58 0.99 0.30 cm 0.59 cm 0.57 1.12
Whole-body ICF 30 0.28 1.00 0.08 L 0.16 L 0.27 0.54
Whole-body ECF 30 0.13 1.00 0.02 L 0.05 L 0.13 0.25
Mean fCSA 26 6.65 0.91 459 µm2 899 µm2 6.59 12.92
MF protein 24 15.60 0.93 38.8 µg/mg∗ 76.0 µg/mg 15.44 30.27
SARCO protein 24 4.38 0.99 7.5 µg/mg 14.6 µg/mg 4.33 8.49
These data are from our laboratory (Haun et al., 2018; Roberts M. D. et al., 2018) demonstrating test-retest reliability statistics for macroscopic, microscopic, and
molecular assessments of hypertrophy. Abbreviations and symbol: n, n-size included in the analysis (college-aged men only); CV, coefficient of variation; ICC, intra-class
correlation coefficient; SEM, standard error of the measurement; DXA LBM, dual x-ray absorptiometry lean body mass; VL thick, ultrasound-assessed vastus lateralis
thickness; mid-thigh circum., mid-thigh circumference measured with a tape measure; ICF, intracellular fluid assessed using bioelectrical impedance spectroscopy; ECF,
extracellular fluid assessed using BIS; mean fCSA, type I and type II (combined) muscle fiber cross-sectional area; MF protein, myofibrillar protein assessed through
biochemical methods; SARCO protein, sarcoplasmic protein assessed through biochemical methods; ∗ , µg/mg indicates µg pf protein per mg of muscle used for the
assay (wet weight).
specific research question prior to clustering as discretization of (1) Although we too have underappreciated measurement
continuous data can potentially result in erroneous conclusions. error previously, we feel a worthwhile future strategy
Often, if explaining variation in hypertrophic responses to in the hypertrophy literature is to consistently report
resistance training is the primary aim via predictors of interest, test-retest reliability statistics and/or calculated
researchers would likely be better served to include all subjects standard errors of measurements to better understand
and their raw, continuous scores in the analysis for greater changes more associated with training, nutritional, or
statistical power. supplementation interventions. By understanding the
Finally, calculating the test-retest reliability of hypertrophic expected error of measurement with certain confidence,
assessments to establish standard errors of measurement is also changes in molecular, microscopic, and macroscopic
a powerful strategy to more confidently conclude if hypertrophy assessments of muscle hypertrophy can be more
occurred and direct further exploration. Conceptually, changes accurately surmised with improved confidence.
beyond calculated measurement error can allow researchers to (2) If molecular levels of measurement are not directly
infer that the specific construct being assessed by the employed assessed, researchers should report the results of the
technique changed beyond the error of the measurement, measurement itself clearly stating the outcome measure
regardless of the extent. Often, test-retest reliability is unreported, in its associated unit instead of invoking the general term
and depending on the calculated error of measurement, changes hypertrophy alone. For example: “According to DXA,
within various ranges after resistance training interventions lower extremity lean mass increased by 1 kg.” rather than
may be better explained by measurement error rather than “According to DXA, muscle hypertrophy occurred.”
true variation in the construct being assessed due to the (3) Future work can help clarify the specific type of skeletal
research intervention. As an example, reliability statistics from muscle hypertrophy occurring from an intervention by
our laboratory for macroscopic, microscopic, and molecular assessing connective tissue, myofibrillar, and sarcoplasmic
assessments of hypertrophy are shown in Table 3. Both absolute fractions of muscle.
values (expressed in the unit of the measurement) and relative (4) Finally, investigators should choose and justify muscle
values (expressed as percentages of the measurement) can be used size assessments based on that which best answers their
to construct confidence intervals beyond which changes scores research question. Assessments on different scales are not
may be more associated with factors other than measurement inherently better or worse, but they are different insofar as
error. Importantly, even the calculation of measurement error their construct validity.
possesses a certain degree of confidence, and this is not posited
to negate changes within the calculated range of error, as these
changes could still be real. Nevertheless, the combination of the CONCLUSION
calculated measurement error and the measured change itself
provide more information to the researcher, and reader, and Assessing the macroscopic, microscopic, and molecular
can improve the interpretation and presentation of data. Ideally, adaptations to resistance training has been a widespread research
in the case of a well-controlled resistance training intervention, goal for exercise physiologists since the 19th century. Given the
hypertrophic outcomes could be better associated with the effects current knowledge-gap regarding the ultrastructural adaptations
of training rather than measurement error itself. to resistance training, we hope that future research will better
To summarize, we propose the following strategies characterize the biochemical and ultrastructural underpinnings
for consideration: of skeletal muscle hypertrophy. While different assessment

techniques seem to disagree with one another, we posit architect of this work. All co-authors substantially contributed,
that this conundrum provides tremendous opportunity for edited and approved the final version of this manuscript.
future researchers to build upon current methods or generate
newer and more valid methods to better assess skeletal
muscle hypertrophy. FUNDING
Work discussed herein from the laboratory of MR was
DATA AVAILABILITY supported through contracts from ImpediMed, Inc. (Carlsbad,
CA, United States), Renaissance Periodization (Charlotte, NC,
Publicly available datasets were analyzed in this study. This data United States) as well as donations from Lockwood, LLC
can be found at https://peerj.com/articles/5338/#supp-1. (Draper, UT, United States), Hilmar Ingredients (Irvine, CA,
United States), BioNutritional Research Group (Irvine, CA,
United States), and JW Nutritional (Dallas, TX, United States).
AUTHOR CONTRIBUTIONS
CH and MR conceived the discussed topic based on a symposium ACKNOWLEDGMENTS
lecture delivered by MR at the 2018 World Congress of Muscle
Hypertrophy at the American College of Sports Medicine Annual We would like to thank participants who took part in past studies
Meeting (Minneapolis, MN, United States). CH was the primary from laboratories that are discussed in this review.
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A Critical Evaluation of The Biological Construct

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published: 12 March 2019

A Critical Evaluation of the Biological

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INTRODUCTION in sarcoplasmic volume [i.e., muscle intracellular fluid (ICF)],

First, what is meant by hypertrophy: increase in girth of a limb,

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which further separates muscle into fascicular bundles of

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resistance training in human fibers. Conversely, and as suggested Macroscopic Assessment

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Author/Year Subject and group description Methods Outcomes

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Author/Year Subject and group description Methods Outcomes

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Author/Year Subject and group description Methods Outcomes

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1 DXA 1 VL 1 mean 1 MF 1 SARCO 1 myosin

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DXA LBM 10 0.95 1.00 0.47 kg 0.92 kg 0.92 1.81

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