Impact of Muscle Temperature on Muscle Growth and Breakdown: Cooling During Resistance Training
Phase: NA
Conditions: Exercise Training
Sponsor: University of Nebraska

Study Protocol and Statistical Analysis Plan 
 
Title: Impact of muscle temperature on muscle growth and breakdown: Cooling during resistance 
training 
 
NCT05302791   
Unique Protocol ID 0072-22-FB 
Date: 19 Apr 2022 
 
 
 

Abstract 
The purpose of this study is to examine the effect of human skeletal muscle temperature during resistance 
exercise on myogenic and proteolytic signaling. Study participants will be eligible if they are male or female 
aged 19-45, recreationally active, and considered 'low risk' for activity participation. Participants will perform 
bilateral resistance exercise bouts at an intensity that should stimulate a muscle growth response in the Vastus 
Lateralis. During the resistance exercise, the participants will receive a cold (10°C) intervention on the 
experimental limb, while receiving a neutral temperature intervention (22°C) on the control limb. Skin and 
intramuscular temperatures, local blood flow, and skeletal muscle biopsies will be collected from each leg pre-, 
immediately post, and following the 3-hour recovery to evaluate myogenic and proteolytic related gene and 
protein expression. 
 
Purpose and Rationale 
 
To test the hypotheses that skeletal muscle cooling will inhibit the cell signaling response associated 
with muscle growth and that skeletal muscle cooling will inhibit the resistance exercise induced 
skeletal muscle growth response.  
 
Maintaining an adequate muscle mass is critical for health and athletic performance. The mechanisms 
regulating muscle mass are widely studied and are of interest to a diverse group of people, including 
athletes, strength and conditioning coaches, athletic trainers, and health care professionals. Skeletal 
muscle is a highly plastic tissue able to adapt to changes in response to various stimuli, such as 
mechanical stress, physical activity, nutrients, and growth factors (7). Increased load on muscle results in 
an increase in its mass or hypertrophy, whereas unloading or disuse leads to a decrease in mass or 
atrophy. Based on the currently known mechanisms of maintaining and increasing skeletal muscle mass, 
resistance training is considered to be a powerful stimulus of muscle hypertrophy. It has been 
consistently demonstrated (12-15) that a single bout of resistance exercise is sufficient to enhance 
protein synthesis, which in turn may stimulate myogenic pathways in skeletal muscle. Furthermore, 
resistance exercise induces translational changes in the muscle, which may serve as the basic regulatory 
mechanism in hypertrophy (16). Protein synthesis and protein breakdown are regulated through 
multiple signaling pathways, including the Akt/mTORC1 pathway. In this pathway, Akt activation by 
stimuli, such as mechanical stress can lead to the activation of mammalian target of rapamycin (mTOR). 
mTOR is a serine/threonine kinase that plays a key role in various biosynthetic processes, including 
protein synthesis, cell growth, and cell proliferation (17-20). During growth factor and nutrient 
sufficiency, mTORC1 phosphorylates the translational regulator p70 ribosomal protein S6 kinase 1 
(S6K1). Phosphorylation activates p70 S6K1 and allows it to phosphorylate the S6 ribosomal protein, 
directly leading to protein synthesis at the ribosome (21). p70 S6K1 phosphorylation is a good acute 
marker of muscle protein synthesis, as it is highly positively correlated with increased muscle mass 
following six weeks of electrical stimulation in rodent hindlimbs (22) and 12 weeks of resistance training 
in humans (23). In a non-phosphorylated state, 4E-BP1 binds to eukaryotic translation initiation factor 4 
(EIF4) and inhibits its ability to bind the 40S ribosomal subunit. Phosphorylation of 4E-BP1 leads to its 
dissociation from EIF4, resulting in the 40S ribosomal subunit activating and initiating mRNA translation 
(24). In addition to regulating protein synthesis, mTORC1-mediated phosphorylation of S6K1 and 4EBP 
also promotes cell growth and cell cycle progression (25, 26).  
 

Even though resistance exercise is a powerful stimulator of muscle synthesis, it also inevitably stimulates 
protein degradation (12). Maintenance of skeletal muscle depends upon the balance of protein 
synthesis and degradation, and it plays a crucial role in the remodeling of muscle tissue. Muscle protein 
degradation is modulated by forkhead box O3 (FOXO3a), a transcription factor responsible for the 
expression of proteolytic genes, mainly atrogin-1 and MuRF1 (27, 28). FOXO3a is targeted for 
phosphorylation by several protein kinases, including AKT. When phosphorylated, FOXO3a is 
translocated out of the nucleus, and cannot exert its pro-transcriptional effect on aforementioned 
proteolytic genes (29).  
 
Individuals with compromised muscle mass appear to have a similar response to resistance exercise 
compared to healthy controls based on mRNA expression of muscle regulatory factors (30). However, 
there appears to be a lower level of muscle regulatory factor protein accumulation in aged muscle tissue 
following resistance exercise (31), as well as a lower capacity for hypertrophy in those with muscle 
wasting (31-33). It has been demonstrated that individuals with compromised muscle mass have a 
similar myogenic response at the transcriptional level, compared to healthy controls (30). However, 
there appears to be a lower level of protein accumulation in aging individuals, as well as a lower 
hypertrophic response to resistance exercise in individuals with muscle wasting. These observations lead 
to a conclusion that the overall limitation in myogenic response may result from differences in 
translational and post translational signaling. These findings indicate that these individuals may have a 
limited overall myogenic response to contractile stimuli, possibly due to differences in translational and 
post-translational signaling. These findings highlight the need for development of novel therapies to 
treat the loss of muscle mass and/or increase hypertrophy above and beyond traditional resistance 
training. These exploratory studies propose the novel use of local muscle temperature manipulation to 
augment resistance exercise-induced myogenesis in humans. Previously, altered temperature resulted in 
stimulation of Akt/mTOR signaling in rodents and human cell cultures and downregulated the activity of 
FOXO3a (7, 34, 35), however the responses in exercising human muscle are currently unknown and are 
to be explored in the proposed study. Furthermore, a more comprehensive view of myogenic and 
proteolytic processes following resistance exercise will be examined through the utilization of 
translational and post-translational markers. 
 
The investigator’s laboratory has recently published a proof-of-concept study that investigated the 
human myogenic and proteolytic transcriptional response to resistance exercise when one leg was 
heated, and the other leg was cooled (5). The results from this study indicate that the exercise-induced 
response was different between the heated and cooled leg, in favor of the heated leg. With this initial 
study design, it cannot be determined if heating produced a favorable response or if cooling produced a 
inhibitory response. Furthermore, it is unsure the impact of temperature independent of exercise. The 
currently proposed studies will allow these questions to be answered with greater mechanistic 
resolution. There are few human studies that have addressed this issue. However, these studies do not 
lead to the same conclusions. No statistical benefit in muscle hypertrophy or strength was observed 
with local muscle heating after 12 weeks of resistance training (Stadnyk 2018). It should be noted that 
the difference in muscle temperature between experimental and control conditions was only 
~1°C. Using a similar design Goto et al (2007) demonstrated an increase in muscle hypertrophy and 
strength, although this observation may be due the non-dominant limb serving as the experimental 
(heating) condition. Unfortunately, these previous studies did not measure any molecular aspects that 
could have allowed more clear interpretation of the data and assessment of the protocols used nor did 
they investigate the impact of heat alone. To our knowledge only one study has investigated the 
resistance exercise mTOR signaling response in humans. This study indicates that heat enhances mTOR 
signaling after resistance exercise in human skeletal muscle (Kakigi 2011). Unfortunately, this study used 

microwave therapy to increase the muscle temperature much quicker and greater than typical external 
heat modalities. The issue with the use of microwave therapy is that this modality may have other non-
thermal effects on cell membrane integrity (Rougier 2014) which in turn could activate the mTOR 
pathway. More clear rationale for the use of heat to enhance muscle growth comes from rodent and 
culture models. Heat stress appears to activate the Akt/mTOR pathway (Yoshihara, 2013) and attenuate 
muscle atrophy (Naito, 2000) in rats. 
 
The use of cold-water immersion has become a popular post-exercise recovery strategy despite a lack of 
evidence of its efficacy. Indeed, much of the data available of the human skeletal muscle response to 
local cold has used a cold-water immersion protocol. Strength training adaptations appear to be blunted 
when incorporating cold water immersion (36), which may be due to suppressed ribosome biogenesis 
(37) and anabolic signaling (38). Low cell culture temperature inhibits myogenic differentiation (39), and 
anabolic response (10, 40). These results are not without controversy as other applied studies and 
subsequent meta-analysis have shown a mild benefit of cold interventions (41). The current studies will 
help understand the disconnect between applied human model studies and mechanistic studies in 
rodents and cell culture by implementing molecular cell-signaling data into our human model. 
 
Investigator’s previous work and the foundational studies presented above will allow us to take a 
strategic approach and build off the previous work. We will take applied study design cues from the 
previous human studies and incorporate mechanistic components from rodent and culture studies. 
Furthermore, the studies proposed here will be able to determine the potential separate effects of 
temperature and those of effects of temperature only observable in the presence of exercise. 
Participants 
12 participants, age 19 to 45, will need to complete the study in order to achieve the scientific objectives of the 
research. Keeping an age range of 19-45 ensures that the participants will be relatively homogenous in terms of 
overall health and thus not present added risk. 
Research Plan 
A total of 2 visits to the laboratory will be required following the signing of informed consent. Given participant 
consent, photographs may be taken during the trials to document the research and use in future presentations. 
Initial Visit (~1.5-2 hours): 
Participants will complete a risk stratification form based on ACSM guidelines and must be considered low risk 
in order to participate in this study. Height and weight will be measured using a medical scale. Body fat will be 
assessed with hydrostatic weighing using an electronic load cell-based system (Exertech, Dresbach, MN) 
correcting for residual lung volume, or BIA body composition analyzer (InBodyUSA, Cerritos, CA) if needed. 
Participants will then perform a Repetition Max Test for bilateral seated leg press and seated leg extension 
using the following protocol on isotonic exercise machines at a weight designed to create failure between 8 and 
12 repetitions. Following a short warm-up on a cycle ergometer, the participants proceed with a bilateral leg 
press warm-up consisting of one set of ten repetitions at a self-determined light load, then one set of 5 
repetition at a self- determined moderate load, and one set of 1 repetition at a predicted 12 rep maximum as 
predicted in relation to their Fat Free Mass. During the warm-up participants will be coached on form and 
observed for their ability to complete the repetition max test. Weight for the test will be altered to suit the 
participants’ ability to complete approximately 12 repetitions. Once the desired weight is determined, 

participants will be asked to complete as many repetitions as possible until the participant can no longer 
complete a repetition using a full range of motion. 
An identical testing protocol is then performed for the leg extension. This data will be used to estimate a 12RM 
workload, that would be appropriate for hypertrophic growth using the Watham equation (46). 
Experimental Visit (~6.5-8 hours): 
Participants will be instructed to arrive to the lab having fasted overnight, and they will be asked to avoid 
strenuous activity, alcohol consumption, tobacco use, and drug use for the 24-hour period leading up to 
experimental trial. 
Participants will complete the following experimental protocol briefly described: consume a small meal, a pre-
exercise muscle biopsy, precool for 30 minutes, complete the exercise intervention, immediately post-exercise 
biopsy, 4-hour recovery with cooling, and post- recovery biopsy. At three time points (pre-exercise, 
immediately post-exercise, and post-4-hour recovery), skin temperature, muscle temperature, blood flow, and 
muscle tissue biopsies will be taken. 
Details are as followed: 
Meal 
When participants arrive, a consistent meal will be provided to standardize dietary intake. Food choices will be 
given to participants to create a meal between 700-800 calories, containing approximately 25-35 g of protein, 
75-85 g of carbohydrates, and 35-45 g of fat. These foods will be made-up of commercially available pre-
packaged foods. They will have approximately 30 minutes to consume their meal. 
Cooling protocol 
Participants will have Game Ready Med 4 elite (https://gameready.com/med-4-elite-contrast- therapy-unit/.) 
thigh wraps around each thigh. This system circulates cooled liquid through a wrap specifically fitted for 
different body segments. One thigh will be cooled while the other thigh will be wrapped with the cuff, but not 
have any liquid circulating through it. These wraps do provide slight compression and thus it is important to 
apply the wraps to both legs. Each leg is assigned to either cold experimental group or control, will be 
randomized and counter-balanced between participants. The liquid temperature circulating through the 
thermal wraps will be set to 10°C. This temperature has been shown in investigator’s previous works to not 
injure the participants’ skin. The thermal wraps will be worn during a 30-minute cool down period prior to the 
exercise bout, during the exercise intervention, and during a 4-hour recovery period. 
Exercise Intervention 
The participants will complete an intervention designed to stimulate the muscle growth signaling pathway 
(hypertrophy) in the Vastus Lateralis (outer thigh), via bilateral seated leg press and bilateral seated leg 
extension on isotonic exercise machines. Participants will perform four sets of bilateral leg press exercises at 
the weight determined to be their 12 RM from the repetition max test. This should allow completion to failure 
of 8-12 repetitions. So, if more than 12 reps are able to be completed, weight will be added to the resistance 
for the subsequent sets. Conversely, if less than 8 reps lead to failure, the weight should be reduced for the 
subsequent sets. Constant dialogue with participants will give investigators the ability to monitor set weights to 
maximize a hypertrophic response. Participants will be allowed 2 minutes of rest between each set. Upon 

completion of 4 sets, the participants will be given 5 minutes of rest prior to repeating this protocol for bilateral 
leg extensions. 
Biopsies 
A total of 6 muscle biopsies (1 per leg) will be obtained from the vastus lateralis (outer thigh) muscle. One from 
each leg for each of the three collection time-points, pre-exercise, immediately post-exercise, and post-4-hour 
recovery. The PI will conduct these biopsies, having successfully completed over 600 biopsies without any 
adverse events. Briefly, the area above the vastus lateralis muscle belly will be shaved and cleaned with 
alcohol. 1% xylocaine is then injected under the skin with a 25 ga hypodermic needle. The Xylocaine will be 
ordered with a prescription provided by the participating physician. 
The area is then further disinfected with betadine, and a sterile fenestrated drape is placed over the biopsy 
site. Using sterile techniques, a small incision is made through the skin (~5mm) and the Bergstrom biopsy 
needle is inserted through the incision into the belly of the muscle. The Bergstrom needle then takes a small 
clip of muscle (~50mg) and is removed from the muscle and stored in a chemical stabilizer at -30ºC. Slight 
pressure is held over the incision for approximately 2 minutes and is then closed using steri-strips and a band-
aid with antibiotic ointment. The leg is further dressed with a compression bandage. 
Participants are all instructed how to care for the wound and given the investigators contact information in 
case they have further questions. 
Intramuscular Temperature 
Using the incision from the muscle biopsy, a hypodermic (~26ga) thermocouple (Physitemp Instruments 
LLC, Clifton, NJ) that is smaller than the needle used to inject the xylocaine will be inserted into the belly 
of the muscle to measure intramuscular temperature. The thermocouple readings stabilize in 
approximately 3 seconds and the thermocouple is then removed. Under normal circumstances, the 
sensation of this temperature reading is similar to an intramuscular injection. However, with the use of 
the biopsy incision as the insertion point there is very little sensation associated with this measure. 
Recordings for intramuscular temperature will be collected during the biopsy process following the 4-
hour temperature intervention.  
 
Skin Temperature  
Skin Temperature will be measured on the surface of each thigh using a skin thermistor (Physitemp 
Instruments LLC, Clifton, NJ), infrared digital thermometer, and/or using an infrared thermal camera. 
Measurements will be taken at the same time as the intramuscular. The thermistor is simply placed on 
the skin for approximately 3 seconds and is then recorded. The infrared digital thermometer can 
measure and record surface temperature from a distance of 12 inches. The infrared thermal camera can 
measure and record surface temperatures for a field of multiple surfaces.  
 
Gene Expression Analysis  
The muscle tissue collected will be processed and analyzed for transcriptional changes of genes 
associated with muscle growth and breakdown. Muscle mRNA content will be evaluated using real-time 
reverse transcription quantitative polymerase chain reaction (real- time RT-qPCR). Quantification of 
mRNA for genes of interest will be computed using the 2-CT method and compared to stable 
reference genes. Probes and Primers that will target the specific gene sequences will be attained from 
Integrated DNA Technologies (IDT, Coralville, IA).  
 

Protein Expression Analysis  
The muscle tissue collected will be processed and analyzed for proteins associated with muscle growth and 
breakdown. Standard western blotting protocols will be optimized and used to evaluate the muscle growth 
signaling cascade, using antibodies specific to the proteins of interest and normalized to total protein. 
 
Statistical Analysis 
Recruitment 
Previous exercise research suggests that the difference in response of matched pairs is normally 
distributed with a standard deviation of 0.86. If the true difference of the mean response of matched 
pairs is 0.75, we will need to complete 12 experimental trials for each condition to be able to reject the 
null hypothesis that this response difference is zero with a power of 0.8. The Type I error probability 
associated with this test of the null hypothesis is 0.05.  
 
Interventions 
A repeated measures two-way ANOVA will be used for the dependent variables. If the F-ratio values are found 
to be significant then a Fishers protected Least Significant Difference post hoc will be performed to evaluate 
where the significance occurs. The probability of type I error must be less than 5% in order to be considered 
significant (p <0.05). All Statistical data will be analyzed using the Statistical Package for Social Sciences 
software (SPSS 24.0)