Review Article

Basic Models Modeling Resistance Training: An Update for Basic Scientists

Interested in Study Skeletal Muscle Hypertrophy
Jason Cholewa2# Lucas Guimares-Ferreira3#, Tamiris da Silva Teixeira1, Marshall Alan Naimo4, XIA
Zhi5,6, Rafaele Bis Dal Ponte de S1, Alice Lodetti1, Mayara Quadros Cardozo1, Nelo Eidy Zanchi1*
1- Postgraduate Program in Health Sciences, Health Sciences Unit, Universidade do Extremo Sul Catarinense,
Cricima/SC, Brazil.
2- Department of Kinesiology Recreation and Sport Studies, Coastal Carolina University, Conway, SC, USA.
3- Laboratory of Experimental Physiology and Biochemistry, Center of Physical Education and Sports, Federal
University of Espirito Santo, Vitria/ES, Brazil.
4- Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, USA.
5- Exercise Physiology and Biochemistry Laboratory, College of Physical Education, Jinggangshan University,
Ji'an,Jiangxi, PR China.
6- Exercise Physiology Laboratory, Department of Exercise Physiology, Beijing Sport University, Beijing, PR China.
Running head: Basic models modeling resistance training
Keywords: resistance training, experimental models, skeletal muscle hypertrophy
# Dr. Jason Cholewa and Dr. Lucas Guimares-Ferreira contributed equally
* Corresponding author:
Nelo Eidy Zanchi
Email:neloz@ig.com.br
Av. Universitria, 1105 - Bairro Universitrio
C.P. 3167 | CEP: 88806-000
Cricima / Santa Catarina
Phone: +55 48 3431-2500
Fax: +55 48 3431-2750

This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: [10.1002/jcp.24542]

Received 12 December 2013; Revised 14 December 2013; Accepted 16 December 2013

Journal of Cellular Physiology
2013 Wiley Periodicals, Inc.
DOI 10.1002/jcp.24542

Abstract
Human muscle hypertrophy brought about by voluntary exercise in laboratorial conditions is the most
common way to study resistance exercise training, especially because of its reliability, stimulus
control and easy application to resistance training exercise sessions at fitness centers. However,
because of the complexity of blood factors and organs involved, invasive data is difficult to obtain in
human exercise training studies due to the integration of several organs, including adipose tissue,
liver, brain and skeletal muscle. In contrast, studying skeletal muscle remodeling in animal models
are easier to perform as the organs can be easily obtained after euthanasia; however, not all models
of resistance training in animals displays a robust capacity to hypertrophy the desired muscle.
Moreover, some models of resistance training rely on voluntary effort, which complicates the results
observed when animal models are employed since voluntary capacity is something theoretically
impossible to measure in rodents. With this information in mind, we will review the modalities used to
simulate resistance training in animals in order to present to investigators the benefits and risks of
different animal models capable to provoke skeletal muscle hypertrophy. Our second objective is to
help investigators analyze and select the experimental resistance training model that best promotes
the research question and desired endpoints.

Introduction
In humans early training gains in muscle strength have been regarded as the result of both neural
and musculature adaptations. Over the last half-decade several animal training models have been
developed as a way to increase both force output and mass (hypertrophy) in the exercised muscle.
Contrary to the increases in maximal oxygen consumption observed in animals with aerobic training
using a treadmill, measurements of maximal and submaximal force capacity in vivo are complicated
by several factors, including voluntary capacity to perform resistance training, non-voluntary
electrical-based training under anesthesia, surgical manipulation of muscles involved in the
hypertrophic response, and the utilization of positive or negative reward to stimulate the animals to
perform the exercise. Thus, the greatest motivation for an animal to produce maximal capacity
voluntary muscular force in classic operant models is via direct electrical stimulation to the brain,
which is virtually impossible to perform in subsequent experiments with the same animal (Olds and
Milner 1954).
Pain avoidance has been demonstrated to be a greater stimulus than food or water reward (Miller
1951). According to Timson (1990), the animal will perform a task only until the effort involved in the
task performance exceeds its desire for the stimulus. Thus, a model employing starvation as the main
stimulus will motivate the animal to exert only 50-60% of its maximal voluntary capacity, which will
then negatively affect muscular hypertrophy capacity either due to lack of overload or nutrition.
Therefore, we will first review animal models employing non-voluntary maximal capacity force
production as a way to induce hypertrophy, and then discuss new methods involving voluntary
models. A summary of results of the models reviewed is available in Tables 1 and 2.

Non voluntary non electric exercise-induced enlargement in animal models.

One of the first methods to induce skeletal muscle hypertrophy was developed by Thomsen and Luco
(1944) whereby a passive stretch applied to immobilized joints places longitudinal tension upon the
muscle (Alway et al. 1989) (Fig. 1F). Utilizing this model of overload Aoki et al. (2006) reported an
increase in sarcomeres in series leading to an elongation of the target muscle. The application of
rapamycin was demonstrated to robustly suppress this response, suggesting the mammalian target
of rapamycin (mTOR) pathway is involved in the longitudinal hypertrophy induced by joint
immobilization. This model of overload may be appropriate to study skeletal muscle remodeling as a
result of stretch overload or joint immobilization; however, resistance training in humans requires
dynamic tension generation, resulting in a force overload, and leading to the synthesis of additional
sarcomeres in series. Therefore, future investigators sought to develop methods that more closely
modeled resistance training.

Goldberg et al. (1968) developed an effective non-voluntary non-electrically stimulated model (Fig.
1A) to induce skeletal muscle hypertrophy through synergistic ablation (surgical removal of a
synergistic muscle, most often the gastrocnemius calcaneus portion, generating overload and muscle
hypertrophy of the soleus and plantaris muscle). Although the use of this model to mimic the effects
of human strength training has been highly criticized due to the surgical procedures (Taylor and
Wilkinson 1986), McCarthy et al. (2011) demonstrated no differences in muscle hypertrophy between
mice with genetic satellite cell depletion and non-depleted controls with 2 weeks of synergistic
ablation overload. Given the similar significant improvements in muscle hypertrophy in both groups,
synergistic ablation remains an effective method to study cellular signaling pathways leading to acute
skeletal muscle hypertrophy (Miyazaki and Esser 2009).
On the other hand, because the targeted muscle is exposed to a static stimulus (the animals
bodyweight) the increase in muscle mass occurs most rapidly during the first week of the protocol
and appears to reach a plateau 2 weeks following surgery. Additionally, the animal is under constant
overload every time it moves, compared to separate training sessions used in human resistance
training or other animal models. Thus, synergistic ablation cannot be used in long term studies nor
does it appear compatible with modeling the progressive overload or periodization phases and
nutrition schedules required in human resistance training to induce maximal changes in hypertrophy
and strength.
Tenotomy is a technique where the gastrocnemius tendon is detached and the synergistic muscle is
placed under increased muscle tension (Fig. 1A). Tenotomy appears less effective at inducing
overload and the resultant musculature hypertrophy of the synergist (ex. plantaris) when compared
with surgical ablation (Timson 1990). Although the reason for the difference is not clear, it appears
that the cut tendon is able to reattach when left intact within the muscle fascia. The critiques of
tenotomy are the same as those related to synergistic ablation methods; however the magnitude of
hypertrophy is less and the possibility of the gastrocnemius tendon reattaching the calcaneus tendon.
The use of chronically restricted venous blood flow was first reported by Kawada and Ishii (2005) to
induce skeletal muscle hypertrophy in rats. This model does not involve exercise; rather, blood flow
to the hind limbs is diminished via a surgical intervention. Fourteen days following the operation the
plantaris muscle increased in dry weight by 10% and the concentration of myofibrillar protein
increased by 23%. Additionally, levels of nitric oxide synthase and the muscle insulin like growth
factor-1 (IGF-1) also increased. It is difficult to speculate on the level of difficulty or safety of this
model as a detailed description of the surgery is not completely available in the literature; however,
this model appears to be consistent since Kawada and Ishii (2008) reproduced the results of the first
study and also reported decrements in type I muscle fibers. Although plantaris hypertrophy was
modest compared to synergist ablation, chronic blood flow restriction may be a novel model to study
hypertrophy in animals. When translating the results to human training two questions arise: 1) What

are the effects of chronic blood flow restriction combined with muscular tension? 2) Does blood flow
restriction occurring for longer than 2 weeks compromise the health of the animal or result in a
plateau in muscle hypertrophy? Given that intermittent blood flow restriction under low tension
phosphorylates P70S6K and muscular hypertrophy in humans (Fujita et al. 2007), answering these
questions are essential to evaluating the ability to translate this model to human resistance training.

Non voluntary, electric exercise-induced enlargement in animal models.

Wong and Booth (Wong and Booth 1988) developed a novel non voluntary model to load the hind
limb and induce muscle hypertrophy. In this model the animal is anesthetized, the foot is attached to
an immovable metal plate with adhesive tape, and muscular contraction is stimulated electrically with
joint of the animal starting in a neutral position (Fig. 1E). The ability of this model to induce
hypertrophy and increased muscle fiber cross sectional area is inconsistent and produces only
modest results; however, using a modified model, Baar and Esser (1999) demonstrated P70S6K
phosphorylation and polyribosome formation, which indicates that the Wong and Booth model is
capable of increasing protein synthesis.
Godspink (1999) modified the protocol proposed by Wong and Booth (1988) by loading the limb in a
stretched position (elongation) and allowing for the electrical stimulus to induce a dynamic contraction
(Fig. 1I). This combined model resulted in a greater increase in protein synthesis compared to the
elongation model or isometrically loaded models alone. Moreover, using the combination of
elongation and dynamic overload Godspink demonstrated the activation of a transcript derived from
the IGF-1 local to skeletal muscle, which has been labeled mechano growth factor (MGF). MGF
presents an insert with 52 base pairs in the E domain of the gene, which alters the reading frame of
the 3 end, resulting in satellite cell proliferation/activation following muscle damage, ultimately
leading to muscular repair and hypertrophy (Hill and Goldspink 2003). This model allows the
researcher to apply an identical maximal pulse to generate maximal tetanic force, and thus eliminates
the need to readjust the electrical stimuli. Although the combination of muscular elongation and nonvoluntary contraction may be viable in studying acute increases in protein synthesis, electrical pulses
under anesthesia are difficult to perform, as is the ability to apply a consistent, progressive increase
in electrical stimulation to match an increased load required to induce hypertrophy.

Resistance training (RT) exercise under unloading conditions

Another interesting resistance training model was presented by Haddad et al. (2006) whereby rats
were unloaded via hind limb suspension (HS) to induce muscular atrophy for six days. Animals in the
resistance training group (HST) were trained every other day. Briefly, animals were anesthetized and
stimulation electrodes consisting of Teflon-coated stainless steel wire were introduced into the
subcutaneous region adjacent to the popliteal fossa via 22-gauge hypodermic needles. Wire
placement was lateral and medial of the location of the sciatic nerve allowing for field stimulation of
the nerve. The stimulation wires were then attached to the output poles of a Grass stimulus isolation
unit interfaced with a Grass S8 stimulator. This allowed for the delivery of current to the sciatic nerve
resulting in muscle contraction. The right leg was positioned in a footplate attached to the shaft of a
Cambridge model H ergometer, adjusted to produce maximal isometric tension. Each training bout
consisted of a series of four sets of contractions with 5 min of recovery between sets. Each set
consisted of a series of 10 maximal isometric contractions lasting 2 s each with 20 s of rest in
between contractions. Thus each training session lasted for 27 min, during which the muscle was
activated for a cumulative time of 80 s.
Compared with normal controls Haddad et al. (2006) reported the gastrocnemius of the HS animals
decreased 20%. Although the RT program had a positive effect on maintaining relative muscle weight
at a higher level compared with the HS group (8%), this response may in part have been due to
edema, as total protein concentration was slightly lower (7%,) in the HST compared with the HS
group. This response demonstrates the negative impact of unloading on the hind limb musculature by
illustrating that the myofibril pool was indeed a primary target of the atrophy response. The results of
this study suggest that the process of muscle atrophy is not opposite of muscle hypertrophy, and
demonstrate the inability of isometric based RT to spare muscle protein during unloading. Therefore,
although an isometric model of RT may be appropriate to induce hypertrophy, researchers using
resistance training in animal models of diseases (i.e. dexamethasone-induced diabetes) (Nicastro et
al. 2012a) should consider performing experimental pilot studies with dynamic based contractions
prior to data collection.
On the other hand, Fluckey et al. (2002) demonstrated that dynamic resistance training is capable of
preventing muscle wasting during unloading. In this model, Fluckey et al. developed a modified
version of the human flywheel resistance exercise apparatus so rats could be trained while in hindlimb suspension. This poses a major advantage over the model used in Haddad et al. as the animals
can be trained with dynamic resistance exercise independent of gravity and without being removed
from the cage. Briefly, a rat is tethered via a leather and velcro vest attached to a nylon cord and
spooled around an inertia wheel located on the outside of the resistance exercise apparatus. The rat
is allowed to place its feet on a shock grid suspended at the top of the apparatus (to accommodate

the HS state) and an illumination bar capable is located in the apparatus opposite to the shock grid.
The bar is then illuminated which results in a repetition by the animal. The movement is similar to
squats as performed by humans, as extension occurs at the hip, knee and ankle joints. When
required a shock is applied briefly (<1 s) to stimulate the movement of the animal. In this model the
resistance training protocol consists of 11 exercise sessions over a 4-week period, with two sets of a
maximum of 25 repetitions (or point of failure; i.e. the animal would not respond to the illuminated bar
even with a brief foot shock) for each session.
In a recent study Fluckey et al. (2002) employed a 4-week period of HS with 3 training sessions per
week, each session lasting approximately 15 min (Fig. 1J). In contrast to Haddad et al. (2006) the
flywheel resistance protocol resulted in an increase in soleus mass and body mass compared to HS
alone. Moreover, an increase in MPS and a muscle protein sparing effect of 50% was observed
compared to HS alone, demonstrating the effectiveness of dynamic based resistance training over
isometric based resistance training in a state of unloading. Interestingly, the ELD muscle was not
affected by flywheel resistance training, which arouses muscle recruitment questions utilizing this
resistance training model. It should be noted that the 25 maximal repetitions used in this study did not
result in dramatic muscle hypertrophy, and thus researchers using this protocol to investigate
hypertrophy based research questions should consider increasing the volume to provide adequate
overload in order to stimulate muscle hypertrophy during periods of unloading.

Voluntary exercise-induced enlargement in animal models

Yarasheski et al. (1990) investigated whether heavy-resistance exercise training alters the skeletal
muscle fiber composition of young rats. To model resistance exercise the rats where trained to climb
with weights attached to the rats tail a 40-cm 90 degree mesh incline 20 times/day 5 days/wk for a
food reward. The load was progressively increased from body weight the first 5 days, to 60g on day
6, and then 30g was added every 3 days thereafter (Fig. 1G). The researchers observed a
pronounced change in the muscle phenotype and a change in the muscle mass of trained animals,
but only in the superficial region of the rectus femoris, which contained a greater proportion of type,
IIb fibers compared with the deep region. In contrast, Duncan et al. (1998) trained male Wistar rats (3
weeks old) to climb a 40-cm vertical ladder (4 days/week) while carrying progressively heavier loads
secured to their tails. After 26 weeks of training the rats were capable of lifting up to 800 g or 140% of
their individual body mass for four sets of 12-15 repetitions per session; however, no difference in
body mass or absolute extensor digitorum longus (EDL) and soleus mass was observed between the
trained rats and age-matched sedentary control rats. Absolute and relative heart mass was greater in
trained rats than control rats, and when expressed relative to body mass, the mass of the extensor

EDL and soleus muscles were greater in trained rats than control rats. Despite an increased ability of
the rats to lift progressively heavier loads, this heavy resistance training model did not induce gross
muscle hypertrophy nor did it increased the force-producing capacity of the EDL or soleus muscles.
The discrepancy in results between Duncan et al. (1998) and Yarasheski et al. (1990) was likely due
to the muscles sampled and measured. The soleus is predominantly type I muscle fiber and likely did
not suffer enough overload to induce hypertrophy. We suggest researchers using the ladder climb
model to study hypertrophy or molecular signaling in protein synthesis evaluate samples from
muscles with a higher proportion of type II fibers, such as the rectus femoris or gastrocnemius.
This model of mesh scale was then modified into a second one where Sprague-Dawley rats were
trained to climb a 1.1-m vertical (80 degree incline) ladder with weights secured to their tail
(Hornberger and Farrar 2004). The rats were trained once every 3 days for 8 weeks. Each training
session consisted of 4-9 (6.02 +/- 0.23) climbs requiring 8-12 dynamic movements per climb. Based
on performance, the weight carried during each session was progressively increased. Over the
course of 8 weeks, the maximal amount of weight the rats could carry increased 287% and the
improved training performance was associated with a 23% absolute increase in the weight of the
flexor hallucis longus (FHL), with a concomitant 24% increase in both total and myofibrillar protein.
On the other hand, Scheffer et al. (2012) analyzed oxidative stress in skeletal muscles using a similar
model of climb ladder (43 steps) in 4 different resistance training protocols: Muscular resistance
training: RT consisted of climbing the ladder carrying a load of 10% of body weight, which was
progressively increased to 20%, 30%, 40%, and 50%, 3 to 6 sets with 2-min breaks, and 1215
repetitions. Hypertrophy training: HT consisted of climbing the ladder carrying a load of 25% of body
weight, which was progressively increased to 50%, 75% and 100%, 3 to 6 sets with a 2-min break
and 810 repetitions. Strength training: ST consisted of climbing the ladder carrying a load of 25% of
body weight, which was progressively increased to 50%, 100%, 125%, 150%, 175%, and 200%, 3 to
6 sets with a 2-min break, and 35 repetitions (Fig. 3). After 12 weeks of training on alternate days,
body weight was not different amongst groups and the red portion of the brachioradialis was removed
and oxidative parameters were assessed. Although muscle hypertrophy was not measured, HT
caused an imbalance in oxidative parameters in favor of pro-oxidants, leading to oxidative stress in
skeletal muscle.
In a related study Lee et al. (2004) tested whether adenoviral administration of IGF-I (rats were
injected with recombinant AAV harboring rat IGF-I cDNA (rAAVIGF-I) was capable to increase FHL
muscle mass. Using the ladder climb model (1-m ladder with 2-cm grid steps and inclined at 85), 8
wks of resistance training, a 23.3% increase in muscle mass was observed in the FHL (Fig. 1D). Viral
expression of IGF-I without resistance training produced a 14.8% increase in mass and the combined
interventions produced a 31.8% increase in muscle mass. Therefore, the combination of resistance
training and overexpression of IGF-I induced greater hypertrophy than either treatment alone. These
results suggest that a combination of resistance training and overexpression of IGF-I could be

synergistic and can improve muscle hypertrophy through adenoviral transfections. It must be
remembered that this original finding was revolutionary at that time and generated a lot of knowledge
paving future research. Differences in muscle remodeling following the same regimen used by Lee et
al. (Lee et al. 2004), Hornberger and Farrar (2004), Duncan et al. (1998), and Yarasheski et al.
(1990) could be related to different muscles sampled in each work, the ladder model, such as the
size and number of steps (which differed considerably amongst different the 4 studies), and the
number of sessions per week, load progression, and volume in the protocols. Thus, the ladder model
is a tool capable to induce positive adaptations in muscle hypertrophy; however, minor modifications
to the protocols may greatly affect the results such that the functionality of the model is reduced when
muscular hypertrophy is a major endpoint, thereby reducing the ability to study the effects of genetic
manipulation or ergogenic aids.
To monitor the variance in overload and work performed between groups we suggest measuring
venous lactate and modifying the load appropriately. Scheffer et al. (2012) demonstrated the
effectiveness of this method to equalize the load between groups. Additionally, we suggest
researchers using this model to induce hypertrophy modify the length of the ladder by reducing the
number of steps the animal climbs and increasing the load to more closely mimic human strength
training. As an example Scheffer et al. (2012) employed a hypertrophy protocol of 48 steps with 1.1
cm between steps. Although hypertrophy was not measured, a relationship exists between exerciseinduced oxidative stress and muscle hypertrophy (Wadley 2013), suggesting that sets of less
repetitions may be most effective in inducing hypertrophy. Additionally, this specific hypertrophy
protocol on the ladder may be the most appropriate for evaluating satellite cell activation and
differentiation with resistance training.
Another animal model of voluntary resistance exercise was proposed by Klitgaard et al (1988): rats
were trained to perform a plantar extension in order to obtain a pellet of food (Fig. 1C). The original
protocol was performed in 2 year old rats and after 36 wks of training plantaris muscle mass
increased 24%. On the other hand, utilizing the same protocol but in young rats and for only 13 wks
we observed the plantaris muscle hypertrophied by 13% (Zanchi et al. 2009). Our major finding using
this model was that the Atrogenes (MuRF-1 and Atrogin-1), ubiquitin ligases involved in muscle
proteolysis by the proteasome, decreased only in the trained group, demonstrating the ability of this
model to modulate molecular signaling. Since we didnt measure the degree of muscle protein
synthesis or degradation in the isolated muscles, we cannot speculate on the ability of this model to
impact protein turnover as a whole. There are two factors to consider when using this model: 1) a longer
training period is required for hypertrophy to occur when compared to the synergist ablation or the ladder
model, and 2) this model uses starvation to motivate the animals to perform the plantar extension. This

starvation period poses a major issue when studying physiological responses as it affects both
voluntary work and nutrient status, and is also difficult to apply. Thus the translation of this model to

humans must be interpreted with caution, although several acute studies in humans are performed
under starvation conditions (Fujita et al. 2007).
In 1992, Tamaki et al. (1992) described a weight lifting exercise model designed to induce muscle
hypertrophy in the hind-limb by loading the animal with a canvas jacket attached to the torso and
requiring the animal to perform a squat like exercise (Fig. 1H). The main stimulus was provided by an
electric stimulator linked to the tail of the animal so a punishment stimuli was applied and the animals
performed a squat like exercise of progressively increasing loads within a hypertrophy range (6575% 1 Repetition Maximum - RM). Compared with 60 min of treadmill sprints, acute squat training
resulted in an increase in plasma creatine kinase. When sprint and squat training was carried outfor
12 weeks at 4-5 days/week there was a 12% increase in the plantaris muscle compared with control
animals receiving an electric stimulus; however, there were no significant differences compared to
the sprint training group. Although this model contains a similar biomechanical loading and
movement pattern to human resistance training, its ability to overload the animals and induce muscle
hypertrophy is inferior to other voluntary resistance training models, such as the ladder climb or food
motivated plantar extension proposed by Klitgaard et al. (1988).
In 2003, Wirth et al. (2003) developed a revolutionary model where rats were operantly conditioned to
perform a squat exercise via both reward and punishment (Fig. 1K). Food was restricted and rats
were operantly conditioned with food rewards to enter a vertical tube, insert its head into a weighted
ring (either 70 g or 700 g), lift the ring until its nose interrupted an infrared detector, and then lower
the ring. Load cells measured the external force generated, and displacement transducers measured
the vertical displacement of the ring during each lifting and lowering movement. The apparatus and
training procedures were computer automated. Peak force, velocity, work, and power were calculated
for each movement. Rats in both groups easily acquired the task after 12-15 training sessions
conducted 5 days/wk. The median peak force, work, and power per lift for both concentric and
eccentric were greater for the 700g group. Importantly, 8 weeks of lifting both 70g and 700g 5
sessions per week increased plantaris, soleus, and gastrocnemius mass compared to sedentary
controls; however, dry weight and muscular protein content was not measured, thus it is also possible
these increases may have been partly the result of edema and/or inflammation. These results
demonstrate the utility of quantitating the biomechanics of volitional movements and suggest that the
present model can establish and maintain controlled repetitive movements necessary for studying
injury and adaptation in muscles, tendon, and bone. Moreover, contrary to Tamaki et al (1992) the
absolute weight of the rats was not decreased with this training protocol, suggesting that this positive
operant model combined with histological sampling is a valid protocol to study responses to
resistance training.
Given the potential of the models described by Wirth et al. (2003) and Klitgaard et al. (1988), our
group (Nicastro et al. 2012b) proposed an equipment and system of resistance exercise (RE), based

on squat-type exercise for rodents, with the ability to more precisely control the training variables
proposed in Wirth et al. (2003). In this model we developed an operant conditioning system
composed of sound, scent, light, and feeding devices that optimized resistance exercise performance
by the animal (Fig. 1L). With this system, it was not necessary to tie the animal into the device or
impose chronic fasting or electric shock for the animal to perform the task proposed (muscle
contraction). Furthermore, it was possible to perform muscle function tests in vivo maximal voluntary
strength capacity (MVSC) within the context of the exercise proposed and control variables such as
intensity (percent of MVSC or percent of body weight), volume (sets and repetitions), rest intervals
between sets, and exercise session length. Importantly, sound was the main stimulus given to the
animals as a way to optimize learning and reinforce exercise training. Therefore, despite
experimental limitations, we believe that this RE apparatus is closer to the physiological context
observed in humans. When testing the efficacy of this protocol to counteract the effects of 7 days of
5mg/day dexamethasone (a diabetogenic and proteolytic catabolic hormone) in a common model of
skeletal muscle atrophy, we observed that training attenuated the loss of gross muscle mass and
increased plantaris mass when compared to controls. Additionally, we observed an increase in
MVSC in trained animals, but not controls at the end of the study (Nicastro et al. 2012a),
demonstrating the efficacy of this model to attenuate or even prevent atrophy, and as a reliable
technique to study atrophic disease.

Variables to evaluate when selecting a resistance training model

According to Timson (1990), when using animal models to evaluate muscle enlargement produced by
strength training in humans, three factors must be considered: 1) Muscle recruitment and adaptations
in fiber characteristics; 2) magnitude of muscle enlargement. Given the effects of varying models of
voluntary and non-voluntary resistance loading reported by our team and others, we suggest six
other factors to consider: 3) The degree of nutrition required for a positive reward; 4) Negative reward
(i.e. pain). 5) Time spent conditioning the animal to execute the exercise; 6) Duration required to
obtain muscle remodeling; 7) Muscle voluntary capacity percentage; 8) Resistance training under
atrophic or diseased conditions.
1- Muscle recruitment and adaptations in fiber characteristics: With specific study questions (i.e.:
sarcopenia) type II muscle fiber hypertrophy is more relevant that gross hypertrophy in

preventing the loss of muscle mass and function; however, not all models of training are
capable of overloading all muscle fibers and thus eliciting a substantial degree of hypertrophy
in muscles comprised of predominantly Type II fibers. As an example, the plantaris is a mixed
fiber muscle and its hypertrophy through surgical ablation of the gastrocnemius (a
predominantly type II muscle) may not be appropriate to study the reversion of sarcopenia
compared to a squat based model (Nicastro et al. 2012b).

2- Magnitude of muscle enlargement: Some exercise models are difficult to perform and the

resulting hypertrophy degree is very poor when compared with others. For example the ladder
climb model is capable to generate hypertrophy in the muscles of the lower limbs of the rat;
however, the hypertrophy of the FHL vary considerably in the literature based upon ladder
length, load, and frequency (Duncan et al. 1998; Hornberger and Farrar 2004). In contrast,
synergist ablation results in a robust hypertrophy of the FHL (80% hypertrophy with synergistc
ablation in our group observed by Teixeira et al. 2013, unpublished data), but not the thigh
muscles such as the rectus femoris.

3- The degree of nutrition required for a positive reward: Klitgaard (1988) developed a model
whereby a collar and an acrylic cylinder where the rats where trained to feed through

overextension of hindlimb (plantar extension) to take the pellet and then feed. Utilizing this
model, our group (Zanchi et al. 2009) observed it required approximately 24h of food
restriction to motivate the animals to perform the lift in order to obtain a food pellet.
Additionally, the number of repetitions per day was very limited (16 per day), although we
observed an increase of 13% of muscle mass (plantaris and soleus) compared with paired
feeding control group. Thus, future investigations should consider the effects of models that
require nutrition deprivation to perform exercise when major endpoints include robust
increases in muscle hypertrophy.
4- Negative reward: It is well known recognized that punishment is a stronger stimulus than
reward to induce rodents to perform resistance exercise (Zanchi et al. 2009). However,

sometimes this punishment stimuli is detrimental as the appetite of the animals is reduced due
to the endocrine response involved in the fight or flight (stress) reaction, thus impairing the
muscular and molecular adaptations to a pre-determined stimulus. For example, Tamaki et al.
(1992) demonstrated increases in gastrocnemius and plantaris mass with resistance training
compared to control groups following 12 weeks of training; however, the resistance training
group lost approximately 200g of body mass. Thus, punishment in this model influenced the
endocrine response and diminished the appetite of the trained animals such that limb muscle
hypertrophy was likely compromised.
5- Time spent conditioning the animal to execute the exercise: Through two different voluntary
exercise models, we observed that some rats are capable to learn how to execute a task (resistance
training) very fast whereas others are not. Therefore, every time we conducted a resistance training
protocol we selected for both control and intervention groups only the animals capable to learn very
fast. Based on Klitgaard (1988) and Zanchi et al. (2009) and modifying the model proposed by Wirth et
al (2003), we developed a computerized model using both reward and punishment stimulus to induce
rodents to perform resistance exercise (hindlimb extension) (Nicastro et al. 2012b). Based upon the
results from these models, we suggest researchers employing models of voluntary maximal contraction
spend 14-16 unloaded training sessions spaced every day over two weeks to condition the animals to
perform the resistance training protocol.

6- Duration required to obtain muscle remodeling: In our first model of food rewarded plantar
extension (Zanchi et al. 2009), 3 months of training were required to increase plantaris mass

by 13%. Similarly, Tamaki et al (1992) spent 3 month to induce relative bodyweight adjusted
(muscle weight/body weight) increases in gastrocnemius and plantaris mass of 31.4% and
17.9%, respectively, and absolute increase of 12% in plantaris mass when compared with
controls. In comparison, utilizing electrical simulation Baar and Esser (1999) demonstrated
increases in tibialis anterior and EDL mass of 13.9 and 14%, respectively, when performed
twice a week for 6 weeks. Further research is required to elucidate the optimal combination of
frequency (sessions per week) and volume when designing animal models leading to muscle
hypertrophy. Despite similar increases in absolute plantaris weight, the rats in Tamaki (1992)
were trained 2-3 sessions per week more than those in our study. These results are further
confounded by synergist ablation where the target muscle (usually the plantaris) is chronically
under a load every time the rat moves (similar to a human carrying extra bodyweight, not
taking part in resistance training). Thus, each model has specific characteristics, and based
upon the load (light or heavy) and expected outcomes pilot sessions should be conducted to
determine appropriate frequencies and study durations.
7- Muscle Voluntary Capacity Percentage: It has been well described in the literature that
intensities in the range of 75-85% 1RM are ideal to increase/hypertrophy the muscle mass.
However, utilizing some models it is almost impossible to predict those ideal ranges. For
example, utilizing the ladder model some investigators have employed a maximum effort test
in the ladder model; however, the ladder contains 16-24 steps so the rodent is performing, in
fact, a test of local muscular endurance (i.e.: a 16 RM test) and not a true 1 RM. Predicting the
maximal voluntary power output of a rodent for a given stimulus is also difficult and unreliable.
For example, when we tested the food-reward model developed by Klitgaard (1988) the rats
were unable to perform more than 8 repetitions per workout (Zanchi et al. 2009). A major
concern was whether the rat actually exerted maximal effort, or if it was satisfied by a reduced
food intake. This issue was partially resolved in our more recent model (Nicastro et al. 2012b)
as we observed the rats performed nearly 50 repetitions per session. Moreover, we were able
to measure force developed through a computerized model and compare that to MVSC to
ensure the animals were working the hypertrophy range. Thus, we believe this voluntary
model allows the researcher to more precisely manipulate volume and intensity in the study of
training induced muscular adaptations, specifically hypertrophy and soft tissue remodeling.
8- Resistance training under atrophic or diseased conditions: Several models of atrophy have

been described in the literature; the most commonly used mimics that of spaceflight
(gravitational unloading) via hind limb suspension. Under this condition of atrophy, isometricbased resistance training models capable of producing robust hypertrophy under gravitational
conditions have failed to induce hypertrophy or counteract atrophy (Haddad et al., 2006). It is
possible that this resistance training model may have attenuated the loss of muscle mass

under a gravitational model of atrophy (i.e.: administration of rapamyacin). On the other hand,
it is well established in the literature that dynamic resistance training plus nutritional support
(mainly proteins containing high biological value and leucine) are capable of robustly activating
protein synthesis pathways (Phillips 2011) and counteracting unloading-induced loss of
muscle mass as demonstrated by Fluckey et al. (2002). When considering the catabolic state
as generated by glucocorticoids and diabetes mellitus, we demonstrated that resistance
training is capable of counteracting the loss of muscle mass (Nicastro et al. 2012a). Although
further research in humans is needed to describe the hormonal milieu and muscle activation in
response to daily living activities in humans with atrophic disease, our model provides
researchers with a greater degree of control over the variables involved in resistance training,
and can be used to study the effects of resistance training on atrophy during conditions of
gravitational unloading and glucocorticoid catabolism.
In example of how these factors may affect investigation outcomes, Miyazaki et al. (2011) utilized the
synergistic ablation surgery to investigate the involvement of ERK/MERK pathways and the wellknown Akt/mTOR/P70S6K pathways of protein synthesis initiation in the muscle hypertrophy
phenomena. Using the synergist ablation model of muscle hypertrophy, early and late periods of
muscle adaption were examined. Specifically, they measured these adaptations in a model of form
follows signaling function observing plantaris hypertrophy weight from day 0 to day 10 on a daily
basis. Miyazaki et al. (2011) demonstrated that Akt phosphorylation (Ser 473 or Thr 308) was not
activated until days 2-3, whereas P70S6K (Ser 389, Thr 421/424) and ribosomal protein (RPS6 Ser
235-236) where highly phosphorylated during the entire hypertrophy process. Of note, this delay in
the classical Akt/mTOR activation was accompanied by a rapid and prolonged MEK 1 and 2 (Ser
217/221) activation. The same pattern was observed in ERK 1 and 2 (Thr 221 and 224). Importantly,
this divergence utilizing a well-recognized animal muscle hypertrophy model demonstrated parallel
pathways, one activated early and one later, that both phosphorylated the RP6 protein culminating in
increased protein synthesis. In the early pathway ribosomal kinases phosphorylated the RP6 in the
Ser 235/236 residue. Following the late pathways, RPS6 was phosphorylated in the residues Ser
240/244. This study demonstrates the importance of matching expected outcomes within the
research question to the resistance training model employed. If Miyazaki et al. (2011) had utilized a
model requiring a longer duration of conditioning or training to induce muscle remodeling (i.e.: the
squat model) these novel discoveries may not have been made. Thus, investigators need to be
aware of these particular attributes to resistance training models when investigating molecular
signaling pathways, ergogenic aids, and mechanical tensions and muscle remodeling.

Conclusion

Many important molecular findings regulating skeletal muscle remodeling have been made through
the use of diverse experimental resistance training models. Although a number of models are
capable of inducing muscular hypertrophy, investigators should consider several new variables when
selecting the most appropriate model to answer the research question. For example, investigators
should be aware of the early and late signaling pathways leading to hypertrophy, and chose a model
that activates the appropriate phase. In this same regard, if a large degree of muscular remodeling is
required to answer a research question selecting a hypertrophy model with a weak magnitude will
result in compromised findings.
A major issue facing clinic scientists investigating the medicinal/therapeutic effects of resistance
training is that genetic modifications in animals that are available to basic scientists are not applicable
to the general population. Consequently, a growing emphasis is being placed on investigating the
efficacy and effectiveness of nutritional supplements and ergogenic aids in conjunction with
resistance training. Although the discovery of new signaling pathways has not suddenly changed or
advanced the methods people use to train, overtime these discoveries have led to more effective
training methods, especially when resistance training is used as physiotherapy. As the ability to more
accurately manipulate the variables involved in rodent resistance training models (volume, intensity,
frequency, etc.) improve, scientists will be better able to study the effects of distinct differences in
program design on molecular signaling and hypertrophy. These improvements in model design and
the results obtained can then be used to improve the translation between basic scientific discoveries,
clinical practices, and the application to human health and performance.

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Figure legends
Figure 1: Progression of the Development of Resistance Training Models in Rodents
(1968-2012). A) Surgical ablation; B) Tenotomy; C) Voluntary plantar extension; D) 85
weighted ladder climb; E) non-voluntary hind-limb extension; F) Passive stretch; G) 90
weighted ladder climb; H) Electric stimulated squat; I) Modified non-voluntary hind-limb
extension; J) Modified flywheel with hind-limb suspension; K) Operantly conditioned squat; L)
Modified operantly conditioned squat. M) Jumping submersed in water with overload.
Adapted by the authors.

Table 1. Voluntary muscle hypertrophy models

Author

Year

Yarasheski et al

1990

Duncan et al.

Technical

Size / Height

Angulation

Sessions / wk

Training
period

Series / day

Muscle
hypertrophy
(%)

Progressive lift with loads

attached to the tail using a
mesh

40 cm

90 Degrees

5 days / wk

8 wk

20

Increase in RF
weight

1998

Ladder climbing

40 cm

Vertical

4 days / week

26wk

12 to15

Increase in
EDL and SOL
weights
relative to
body mass
and fibre
hypertrophy

Lee et al.

2004

Ladder climbing + IGF-I

adenovirus

1m

85 Degrees

Every third day

8 wk

8 climbs or
until failure

Increase in
FHL weight

Klitgaard et al

1988

Plantar flexion of ankle joint

In the morning, at
noon, and in the
evening (Monday
and Tuesday,
Thursday
and Friday)

36 wk

30 min of
training 3
times per day

Increase in
SOL and PLA
weights

Zanchi et al.

2009

Plantar flexion of ankle joint

3 Times / Week

12 wk

16

Increase in
PLA weight

Tamaki et al.

1999

"Squat Like Exercise"

4-5 Days / Week

12 wk

65-75 % 1 RM

Hypertrophy of
GAS and PLA
and increase
in the number
of muscle
fibers

Dela Cruz et al.

2012

Jump in a PVC cylinder

containing water

Every two days

5 wk

15 jump
sessions

Increase in
EDL and SOL
CSA

Wirth et al

2003

Operant conditioning
(progressive lift)

8 wk

12 to 15

Increased
performance

Hornberger, T.A

2004

Ladder climbing (with

progressive load attached to
the tail)

1,1 m

80 Degrees

8 wk

Increase in
FHL weigth
and total and
myofibrillar
protein content

Scheffer, et al.

2012

Ladder climbing

43 steps

Alternate days

12wk

3 to 15

Fluckey et ai

2002

Flywheel Resistance
Training

4wk

25

Attenuation of
hindlimb
suspensioninduced
muscle
atrophy in SOL

RF: rectus femoris; EDL: extensor digitorum longus; SOL: soleus; FHL: flexor hallucis longus; PLA: plantaris; GAS: gastrocnemius; CSA: crosssectional rea.

Table 2. Involuntary muscle hypertrophy models

Author

Year

Technical

Period

Estimulus

Muscle
hypertrophy
(%)

Goldberg et al.

1968

Surgical ablation of GAS

6 Days

Walk

Increase in
SOL and
PLA weights

Goldberg et al.

1975

Tenotomy of GAS

14 Days

Walk

Increase in
SOL weight

Wong and Booth

1988

Weight-lifting exercise

16 wk

Electric +
external load

Increase in
GAS we
weight and
protein
content

Baar and Esser

1999

Surgical implantation of
electrodes and electrical
stimulation

6 wk

Electric

Increase in
EDL and TA
wet weights

Goldspink

1999

Stretch Combined With

Eletrical Stimulation of
Anterior Tibialis Muscle
of Adult Rabbit

4 days

Electric

Increase in
TA wet
weight

Kawada and Ishii

2005

Chronic restriction of
blood flow to muscle

2 wk

Venous occlusion

Increases in
PLA dry
weight/ body
weight and
myofibrillar
protein
content

Haddad et al

2006

Electrical stimulation

6 Days

Isometric
contractions

Attenuation
of hindlimb
suspensioninduced
muscle
atrophy in
GAS
(muscle
weight)

EDL: extensor digitorum longus; SOL: soleus; TA: tibialis anterior; flexor hallucis
longus; PLA: plantaris; GAS: gastrocnemius.

Figure 1