Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults
Phase: NA
Conditions: Sarcopenia, Aging, Immune Function
Sponsor: VA Office of Research and Development

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
Page 1 of 43 
Human Subjects Research Protocol 
Title:   
Immune Function and Muscle Adaptations to Resistance Exercise in 
Older Adults 
Funded by:  VA Rehabilitation Research and Development 
Administered by:  Central Arkansas Veterans Healthcare System 
Study Sites: 
Clinical Research Unit 
Research Laboratories 
Central Arkansas Veterans Healthcare System 
Geriatric Research Education and Clinical Center 
2200 Fort Roots Drive  
Bldg 170, Unit 3J, 156 and 157 
North Little Rock, AR 72114 
Central Arkansas Veterans Healthcare System 
4300 West 7th Street 
Research Laboratory GF127 
Little Rock, AR  72205 
University of Arkansas for Medical Sciences 
4301 West Markham 
Biomed I, Rooms 533 and 535 
Little Rock, AR  72205 
Contact Information: 
Richard A Dennis, PhD 
Richard.Dennis2@va.gov 
Office:  501-257-3503 
Cell:  501-960-8024 
Lay Summary:  The loss of muscle mass and strength due to aging leads to serious health problems 
for older adults. Muscle health can be improved by exercise training, but some people improve their 
strength substantially, whereas others improve little. The reason for this variation is unknown. This 
study will investigate whether function of the immune system influences how well people respond to 
exercise.  Older veterans who participate will have their muscle size, strength, and function measured 
periodically for almost a year.  Participants will drink a nutritional supplement or placebo daily and 
complete a 36 session strength training program. Participants will be vaccinated for tetanus and donate 
small amounts of blood and muscle tissue during the study so that immune function can be compared 
to muscle outcomes during training and during a long-term follow-up.  The study results should 
increase our understanding of the negative effects of aging on muscle and will possibly lead to better 
strategies for muscle maintenance and rehabilitation for older adults. 
NCT02261961

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
Page 2 of 43 
 
Key Points of the Supplementation and Exercise Protocol 
• 
Rationale:  Exercise is clearly able to affect immune function.  However, the proposed study will 
attempt to modulate immune function by nutritional supplementation and determine the effects on 
exercise training outcomes in older adults.  
• 
Importance:  Targeting the immune system with nutritional supplementation may be the advantage 
needed for an older veteran to successfully use exercise training to maintain or restore the muscle 
mass, strength, and function that is necessary for personal independence. 
• 
Double-blind randomized placebo-controlled trial:  groups consuming nutritional supplement 
(Muscle Armor) or placebo (Kool-Aid) will be compared to answer four key questions: 
o Do 2-weeks of supplementation improve immune function? 
o Does supplementation during 12-weeks of progressive high-intensity resistance training 
boost adaptations to exercise at the whole body or cellular level? 
o Does continued supplementation for 26-weeks after completion of exercise training promote 
the retention of the gains in muscle size, strength, and function? 
o Do measures of immune function correlate with cellular and whole-body measures of muscle 
health after exercise training and detraining? 
• 
Procedures to be completed by veterans (age 60-80, N=50) 
o Telephone pre-screening with required waiver of HIPAA and permission for verbal consent 
o Enrollment screening after written consent and HIPAA authorization 
o Approximately 50-60 research visits over approximately one year 
o Data collection from CPRS problems and medication lists 
o Orange-flavored supplement or placebo consumption mixed in water twice daily (~42 
weeks) 
o Supervised High-Intensity Resistance Exercise (two sessions prior to training to measure 
response to unaccustomed exercise) 
o Supervised High-Intensity Resistance Exercise Training (36 sessions in ≥ 12 weeks) 
o Post-training follow-up (~26 weeks) 
o Blood draw (9x) 
o Muscle biopsy (5x) 
o Gait and balance testing (4x) 
o TDAP Vaccination (1x) 
o Thigh CT Scan (3x) 
o Compensation (~$1500):  $20 per visit, completion bonus for exercise training ($100) and 
entire study ($300).  $20 bonus if veteran refers another eligible subject to participate.

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
Page 3 of 43 
 
ABSTRACT 
Objective: The study will examine the influence of immune function in older adults on improvement of 
muscle mass, strength, and function by resistance training. The maintenance of those benefits during 
long term follow-up will also be examined. This objective will be accomplished by a double-blind 
randomized placebo-controlled trial of a nutritional supplement (Muscle Armor) which evidence 
suggests can improve immune function, promote muscle growth, and counteract muscle loss. The 
study premise is that aging results in decreased ability of the immune system to respond to stimuli such 
as exercise. The study proposes that the supplement will improve muscle health by promoting a shift in 
immune function of older adults from a pro-inflammatory state towards a state which supports muscle 
growth and maintenance. 
Research Plan: The study will randomize veterans (age 60-80, N=50) to participate in the supplement 
or placebo groups in a three phase study. The phases of participation correspond to the three specific 
aims.  Aim 1 will determine if 2-weeks of supplementation improves immune function. Humoral immune 
function will be assessed as the response to vaccination. Innate immune function will be measured as 
systemic and cellular responses to acute resistance exercise that our previous studies indicate are 
affected by aging. Aim 2 will determine if supplementation during 36 sessions of progressive high-
intensity resistance training boosts improvement in muscle size (CT scan), strength, and function (gait 
and balance).  Muscle adaptations at the cellular levels will also be measured. Aim 3 will determine if 
continued supplementation for 26-weeks after completion of exercise training promotes the retention of 
the gains in muscle size, strength, and function. Multivariable testing will then be used to compare the 
results between Aims 1, 2, and 3 to determine whether or not immune function is correlated with 
muscle adaptation to training or detraining. 
Methods:  Participants will undergo nine blood draws and five muscle biopsies of the vastus lateralis 
over the course of the study so that the effects of the supplement on immune function and cellular 
adaptations to training can be measured. Three of the blood draws will be used to assess the antibody 
response to the tetanus, pertussis, and diphtheria vaccine.  Muscle and blood will be collected before 
and after a bout of exercise conducted before and after the 2-weeks of supplementation prior to 
training. Immune function will be measured using the blood based on pro- and anti-inflammatory 
cytokine levels, the balance between specific T-cell subpopulations, and the proliferative capacity of 
mononuclear cells.  Immune function will be measured in muscle based on macrophage content of 
specific cytokines and growth factors. Our previous study showed that these muscle measures strongly 
correlate with size and strength gain after training. Key signaling pathways including nuclear factor-k B 
and PI3 kinase will also be measured. The fifth biopsy will be collected post-training to measure 
adaptation at the cellular level based on changes in number of satellite cells and myonuclei and fiber 
size. Hypotheses related to these measures will be tested with 80% power to detect at least 0.8 
standard deviations difference in means between the supplement and placebo groups. 
Clinical Relevance:  Exercise is clearly able to affect immune function. However, the proposed study 
will attempt to modulate immune function and determine the effects on exercise outcomes. The study 
will also examine detraining, an important issue for older adults, that is usually omitted from training 
studies. Thus, the study will potentially advance our understanding of the mechanisms of muscle gain 
and loss in older adults, but more importantly, the study will evaluate a nutritional intervention as a 
complement to exercise for supporting muscle health during aging. Targeting the immune system may 
be the advantage needed for an older Veteran to successfully maintain or restore the muscle mass, 
strength, and function that is necessary for personal independence. 
 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
Page 4 of 43 
 
SPECIFIC AIMS 
Age-related decline of the immune and musculoskeletal 
systems leads to serious health problems for older adults.  
The bodily systems are often co-dependent during health 
and dysfunction, but the role of immune function in 
maintaining or improving muscle health during aging is 
unknown.  The relationship between these two systems will 
be examined through the conduct of a randomized trial 
using a nutritional supplement (Muscle Armor, Abbott Labs) 
that is proposed to have beneficial effects on both immune 
function and skeletal muscle.  The trial will determine if the 
supplement is able to improve immune function, boost 
muscle adaptation to resistance training, and support 
retention of the exercise-derived benefits (Figure 1).  The 
supplement ingredients, methylbutyrate, arginine, and 
glutamine (HMB, Arg, and Gln), are used by weight-lifting 
enthusiasts to boost gains from resistance training and 
clinically to counteract muscle wasting.  Thus, an exercise 
trial evaluating Muscle Armor could identify a means to 
make a significant impact on muscle function, health, and 
quality of life for older adults.  However, the mechanisms by 
which this supplement improves muscle health are 
unknown.  Thus, in addition to the potential for direct benefit 
to aging Veterans, conduct of the proposed trial will allow 
the mechanistic relationships between immune function and 
adaptation to exercise training and detraining to be investigated.  
1. Determine the effects of Muscle Armor consumption by older adults on the humoral 
response to vaccination and specific cellular immune responses to an exercise stimulus.  
Our findings indicate that various measures of immune function decrease with aging [1;2].  Aim 1 
will determine if these measures are improved by 2-weeks of nutritional supplementation.  Humoral 
function will be assessed using the tetanus, diphtheria, pertussis vaccine.  Cellular function will be 
assessed using muscle and blood collected before and after a bout of exercise.  Immune cell 
proliferation and phenotype, and levels of cytokines and growth factors and their signaling actions 
will be measured. 
2. Determine if Muscle Armor consumption affects the adaptive responses to resistance 
exercise training at the whole body or cellular level.  Our studies have shown that muscle 
adaption to training varies greatly between older adults [3].  Thus, a nutritional supplement that 
boosts the response could be quite valuable to certain individuals.  Aim 2 will have subjects 
continue supplementation while completing 36 sessions of high-intensity resistance training.  Whole 
body measures of adaptation to resistance training will include changes in muscle strength, cross-
sectional area, and function.  Cellular measures of muscle adaptation will include changes in 
satellite cell and myonuclear numbers, and fiber size. 
3. Determine if Muscle Armor consumption affects the retention of benefits of exercise during 
long-term follow-up.  Training studies of older adults rarely measure post-training maintenance of 
muscle cross-sectional area, strength, and function.  However, this information is highly important.  
The supplement chosen here has shown evidence of ameliorating muscle wasting in chronic 
disease and is being tested for slowing sarcopenia [4;5].  Aim 3 will determine if continuing 
supplementation during a post-training follow-up period (i.e. detraining, 26-weeks) promotes the 
retention of benefits from exercise in older adults.
 
Figure 1.  Randomized trial of a nutritional 
supplement to determine if immune function 
influences muscle health in older adults. 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
Page 5 of 43 
 
BACKGROUND AND SIGNIFICANCE 
1. Muscle loss due to aging leads to serious health problems for older adults.  Effective 
strategies for rehabilitation are critical to the health of older adults since muscle loss decreases 
functional ability and increases risk for falls and fractures, disability, and mortality [6;7].   
2. Resistance training improves muscle mass, strength, and function in older adults but the 
response varies between individuals.  Resistance training can be safely performed with benefit 
to even chronic heart failure and nursing home patients [8], but the magnitude of benefit varies [9].  
This suggests that older adults with less adaptive potential will have greater muscle loss due to 
aging and that rehabilitation will be less effective.  Thus, a better understanding of the effects of 
aging on muscle health may be needed to design interventions that complement resistance training. 
3. The ability of older adults to maintain or improve muscle health may be associated with 
systemic indicators of the effects of aging on immune function.  Age-related decline in 
functions of the immune system results in a weaker response to vaccination [10], diminished ability 
of immune cells to proliferate, and problems that specifically affect muscle.  Acute increases in 
cytokines after exercise initiate muscle repair [11], but with aging inflammation becomes chronic 
and detrimental.  For example, inflammation induces muscle catabolism in rats [12] and is 
associated with sarcopenia and functional decline in humans [13].  Thus, systemic differences in 
immune parameters between older adults may reflect ability to improve or maintain muscle health.   
4. Cells of the immune system residing in muscle play a central role in the developing model of 
muscle adaptation to exercise.  Macrophages transition between three functional phases in 
response to exercise.  Phase 1 is an inflammatory response mediated by cytokines which initiate 
muscle repair and clear debris from damaged myofibers [14].  After this initial cleanup, Phase 2 is a 
down-regulation of inflammation by anti-inflammatory cytokines so that secondary damage does not 
occur.  This allows the muscle to become conducive to growth [15].  In the regenerative Phase 3, 
macrophage arginine metabolism increases and promotes cell growth [16] and growth factors are 
produced to further stimulate myogenesis [17].  As muscle adapts to training, macrophage function 
may then become dedicated to producing growth factors required for hypertrophy.   
5. Evidence suggests that the immune system and macrophages influence muscle mass and 
strength during health and aging.  In animals, extermination of muscle macrophages decreases 
the ability of muscle to hypertrophy and recover from atrophy [18-20].  The effects were related to 
decreased macrophage provision of growth factors, myogenesis, and muscle fiber growth [18;20].  
In humans, muscle mass and strength gain are also associated with markers of myogenesis [21] 
that are diminished in older adults [22].  Inflammation may have contributed to those findings.  In a 
training study of older adults, individuals consuming anti-inflammatory drugs gained significantly 
more muscle mass and strength than those consuming placebo [23].  Our studies also indicate that 
immune function influences the response to exercise.  Muscle size and strength gained by older 
adults from resistance training were strongly correlated with muscle expression of specific cytokines 
and growth factors [3].  Muscle macrophage content in those older adults was also strongly 
correlated with muscle adaptation and growth factor levels.  These findings support the idea that 
consumption of a nutritional supplement that either dampens a negative or boosts a positive effect 
of inflammation and immune function could benefit older adults during exercise training.  
6. Three signaling pathways may be involved in the regulation of macrophage function, 
production of cytokines and growth factors and their effects on muscle cells.  In response to 
pro-inflammatory cytokines and NFkB signaling [24], macrophages may produce cytokines which 
block signaling that increases transcription of ubiquitin ligases which control protein degradation 
and lead to muscle atrophy [25].  However, in response to anti-inflammatory cytokines and STAT3 
signaling, macrophages [24], macrophages produce factors that support tissue growth and block 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
Page 6 of 43 
 
atrophy [26].  Examination of these pathways in human muscle is limited and thus warrants further 
investigation to define their role in the context of aging, exercise, and nutritional supplementation.              
7. A nutritional supplement that affects immune function may improve the ability for older 
adults to benefit from resistance training.  Muscle Armor contains arginine, glutamine, and 
methylbutyrate.  These ingredients slow cachexia and increase muscle mass in both cancer and 
AIDS patients [5;27].  They are also used to increase strength in combination with exercise for 
sports performance [28-30].  Despite this success little is known about the mechanism of action.  
Methylbutyrate alters protein synthesis, but it also enhances immune function in animals [31-33].  
Glutamine and arginine are considered immuno-nutrients that affect immune cell phenotype, 
proliferation, and production of cytokines [34-37].  The study will determine if the effects of the 
supplement on muscle health are mechanistically related to its ability to modulate immune function. 
8. Significance:  Nutritional intervention that targets the immune system may complement 
resistance exercise in helping older adults improve and maintain muscle health.  Exercise is 
clearly affects immune function [38].  However, it is unknown if immune function affects individual 
benefit from exercise because studies have not altered immune function and determined the effects 
on training outcomes.  The proposed study will attempt to do this using a supplement that supports 
muscle health.  The study will also examine detraining, an important issue, that is often omitted 
from exercise studies [39].  Thus, the study has the potential to advance our understanding of the 
mechanisms of muscle gain and loss in older adults, but more importantly, the study will evaluate a 
nutritional intervention as a complement to exercise for improving muscle health in older adults. 
 
PRELIMINARY STUDIES 
The study will determine the relationship between immune function and adaptation to training and 
nutritional supplementation in older adults.  The expectation that improving and maintaining muscle 
size, strength, and function will be correlated with immune function is based on the following findings. 
1. Muscle of older adults, as compared to young adults, has increased expression of cytokines at 
baseline and lacks cytokine induction in response to acute resistance exercise [2]. 
2. Muscle of older adults contains fewer macrophages than muscle of younger adults. The number of 
macrophages that display markers of inflammation or anti-inflammation and growth increase for 
young but not older adults in response to exercise.  Interestingly, macrophages that display the 
marker of anti-inflammation and growth outnumber macrophages displaying the marker of 
inflammation by approximately eight to one [2]. 
3. Muscle of older adults, compared to younger adults, has lower expression of genes associated with 
tissue growth and remodeling at rest and in response to a bout of resistance exercise [40]. 
4. Muscle gene expression associated with inflammation and growth is strongly correlated with muscle 
size and strength gained from training [3].  The number of macrophages in muscle is also correlated 
with size and strength gain, and gene expression suggesting that macrophage provision of 
cytokines, and growth and remodeling factors contributes to muscle adaptation to training.    
5. The effects of aging on muscle macrophages are consistent with the immunosenescence of 
immune cells outside of muscle.  For example, aging decreases the ability of peripheral blood 
mononuclear cells and T-cells to proliferate and respond to pro-inflammatory stimuli [1].   
6. Pilot study findings suggest that the nutritional supplement can dampen systemic markers of 
inflammation and boost the antibody response to the pertussis antigen in older adults. 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
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Finding 1:  Muscle of older adults, as compared to young adults, has increased expression of 
cytokines at baseline and also lacks cytokine induction in response to acute resistance exercise [2].   
Study:  Pro- and anti-inflammatory cytokine mRNA levels were measured in muscle of young (22-41 
yrs) and older (66-80 yrs) males (N=17 each) at rest and 72 hrs after completion of a single session of 
high-intensity resistance exercise targeting the thigh muscles.  At rest, muscle of the older adults 
possessed higher (3.3-fold, P=0.001) the pro-inflammatory cytokine, IL1β, than muscle from the 
younger adults (Figure 2A).  Anti-inflammatory cytokines, IL1RA (P=0.003) and IL10 (P=0.03) were also 
higher in the old than young (not shown).  After exercise, levels of the IL1β increased (~2-fold, 
P=0.006) in the young muscle, but did not change in the old (Figure 2B).  IL10 (P=0.02) also increased 
after exercise but only for the young subjects. 
Conclusions:  Pro- and anti-inflammatory muscle cytokine expression is regulated differently in older 
adults at baseline and in response to exercise.  These results were found for older adults who were 
quite healthy and only ~20% weaker than the young group.  Thus, the overt effects on muscle of this 
altered cytokine expression were not obvious though the results implied that muscle of older adults may 
contain immune cells (i.e. macrophages) in a state of chronic low-grade inflammation. If this is true, 
then it is possible that the chronic inflammation could adversely affect levels of growth factors that 
control muscle hypertrophy which could hinder the adaptive response to resistance exercise training.  
These possibilities were investigated and the results are presented here in Findings 2 through 4. 
A 
B 
 
Figure 2.  IL-1β at rest and in response to exercise is different in muscle from old and 
young adults.  Data for each subject (dots) and medians (bar) are shown.   
 
 
Finding 2:  Muscle of older adults contains fewer macrophages than muscle of younger adults; and in 
both age groups, at baseline and in response to exercise, the majority of macrophages display a 
phenotype consistent with anti-inflammation and growth rather than inflammation [2]. 
Study:  Macrophages in muscle from the young and older males of Finding 1 were counted based on 
markers of inflammation (CD11b) or anti-inflammation and growth (CD163) [16;41;42].  At rest, muscle 
of the older adults contained approximately half as many CD11b+ cells (P=0.003, Figure 3A) as 
younger muscle and tended (P=0.07, Figure 3B) to contain fewer CD163+ cells.  72 hrs after a bout of 
exercise, the numbers of macrophages did not increase in the older adults, but both CD11b+ (55%, 
P=0.04) and CD163+ cells increased in younger adults.  Interestingly, muscle contained 8-times as 
many CD163+ cells as CD11b+ cells (P<0.0001).  
Conclusions:  Muscle of older adults did not contain more macrophages with an inflamed phenotype 
than muscle of young adults.  Muscle macrophages predominantly showed the anti-inflammation and 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
Page 8 of 43 
 
growth phenotype and like cytokines, their numbers only increased after exercise in the young.  
Additional analysis was performed in an attempt to identify factors associated with healing and growth 
which may also impact muscle adaptation in older adults. 
A.  CD11b+ Macrophages / Muscle Fiber 
B.  CD163+ Macrophages / Muscle Fiber 
 
 
Figure 3:  Muscle of older adults contains fewer CD11b+ macrophages and the number does not increase after 
exercise as it does for young adults.  Results for the far more abundant (see y-axes scales) CD163+ 
macrophages were similar though the age-associated difference was only a trend.  Median and inter-quartile 
range are shown.  Significance (P<0.05) between (*) and within (#) groups is shown. 
 
 
Finding 3:  Aging alters muscle expression of genes which function in tissue growth and remodeling 
both at rest and in response to a bout of resistance exercise [40]. 
Study:  The muscle of the young and old adults of Findings 1 and 2 was further analyzed using 
quantitative real-time PCR to measure expression of 100 select mRNAs.  The mRNAs included genes 
that function in cellular stress, signaling, tissue remodeling or growth.  Four mRNAs (IGF1, CNTF, 
MMP2, and IGFBP5) were significantly greater in younger muscle than older muscle both before and 
after exercise (P<0.05).  The median differences ranged from 60% to 230% (shown for IGF1 in Figure 
4A).  Two mRNAs showed a significant (P<0.05) response to exercise.  However, the response was 
found to be significant in only younger adults after statistical correction for multiple comparisons was 
applied.  In younger adults, median TIMP1 levels increased 200% (Figure 4B) and GDF8 levels 
decreased by 50% after exercise (not shown). 
Conclusions:  Gene expression profiling identified a number of muscle mRNAs whose levels are 
affected by aging and acute exercise.  The results included the two most commonly studied regulators 
of muscle mass, IGF1 and GDF8 (also known as myostatin) [43;44] as well as other mRNAs about 
which little is known in the contexts of exercise, aging, and human muscle.  Thus, in addition to the 
factors discussed here in Findings 1 through 3, growth factors such as CNTF and factors that may 
participate in tissue remodeling, namely the metalloproteinase MMP2 and its inhibitor TIMP1, are also 
candidates that require investigation as regulators of muscle mass.  Finding 4 will present the results of 
a training study for older adults that sought to determine the relationship between the molecular and 
cellular variables discussed and gains in muscle size and strength. 
 
 
*******See Next Page for Figure********* 
CD11b+ CELLS / MUSCLE FIBER
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Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
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A.  Influence of Aging on IGF1 mRNA levels 
B.  TIMP1 Response to Exercise 
Figure 4:  Influence of aging and exercise on muscle mRNAs.  A.  IGF1 mRNA is lower in muscle of old 
than young adults before and after exercise (P<0.05). Box plots denote the medians (line), 25th and 75th 
percentiles (boxes), 10th and 90th percentiles (whiskers), and outliers (dots).  B.   TIMP1 mRNA increases 
in muscle of young (P<0.05) but not old adults after exercise.  Median (triangle) and individual subject 
responses (line) are shown.   
 
 
Finding 4:  Muscle expression of genes associated with inflammation, growth, and remodeling is 
strongly correlated in older adults with muscle size and strength gained from resistance training [3]. 
Study:  Older (61-78 yrs) men (N=3) and women (N=5) completed 12-weeks of high-intensity 
progressive resistance training of the thigh muscles.  Biopsies were collected prior to training and 
analyzed by real-time PCR and immunohistochemistry.  Muscle size change range from -1% to +18% 
while strength increase ranged from +8% to +65%.  Strength gain correlated (R > 0.7, P < 0.05) with 
expression of mRNAs encoding cytokines (IL1β, IL10, IL1RA), growth (CNTF, GDF8, IGF1, IGFBP5) 
and remodeling factors (MMP2, TIMP1).  The result is shown for IGF1 in Figure 5A.  Strength gain also 
correlated with the number of CD163+ macrophages in muscle with the exception of one outlier (Figure 
5B).  Similar results were found for muscle size.          
Conclusions:  Macrophages and mRNAs associated with inflammation, growth, and remodeling 
appear to be involved with adaptation to exercise in older adults.  However, these variables could be 
covariates affected by an unknown determinant of muscle adaptation.  The proposed study will allow 
investigation into whether macrophage function or immune function in general influences adaptation to 
resistance training in older adults. 
A.  IGF1 versus Strength Gain 
B.  CD163 Macrophage Number versus Strength Gain 
 
Figure 5.  Relationship between baseline molecular and cellular measures for muscle of older adults and percent strength 
gain for knee extension after 12-weeks of training.  A.  Pearson correlation for IGF1 mRNA and strength gain (triangles = 
males, circles = females).  B.  Relationship between number of muscle macrophages and strength gain.       
RELATIVE EXPRESSION
RELATIVE EXPRESSION
RELATIVE EXPRESSION
RELATIVE EXPRESSION
P=0.001
R=0.94
P=0.001
R=0.94

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
V11, 06-26-2017 
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Findings 5:  Aging decreases the ability of immune cells to respond to stimuli that cause cell 
proliferation and inflammation [1]. 
Study:  The effects of aging on immune cells were examined in two experiments.  Experiment 1 
compared the T-Cell response to a pro-inflammatory stimulus using blood of young (N=5, 21-30 yrs) 
and old (N=5, 65-80 yrs) donors [45].  Cells were treated with TNFα and NFkB binding to its promoter 
was measured.  Binding was lower (P<0.05, 250% versus 340%) in older than younger adults (Figure 
6A).  Experiment 2 examined the ability of peripheral blood mononuclear cells (PBMC) to proliferate in 
response to a phorbol ester and calcium ionophore.  After 5 and 8 days of treatment respectively, 84% 
and 92% of young cells had undergone at least one cell division (Figure 6B); whereas only 52% and 
63% of older cells had divided (not shown). 
Conclusions:  Immunosenescence appears to universally affect mononuclear cells, T-cells, and 
muscle macrophages by diminishing ability to proliferate and respond to pro-inflammatory and exercise 
stimuli.  The proposed study will determine the extent to which immune function external to muscle 
reflects the ability of muscle macrophages to respond to exercise or the ability of older adults to benefit 
from exercise training.   
A. T-Cell NFkB Binding Activity 
B.  Mononuclear Cell Proliferative Capacity 
 
 
Figure 6.  Effects of aging on immune cell function.  A. The NFkB promoter binding assay was used to 
compare the response to a pro-inflammatory stimulus between old and young T-Cells.  B.  Peripheral blood 
mononuclear cells from older adults show less of an ability to proliferate than cells from younger adults in 
response to a pro-inflammatory stimulus.  
 
 
Findings 6 and 7: Pilot study results support the idea that the nutritional supplement, Muscle Armor, is 
able to decrease systemic inflammation and improve the response to vaccination in older adults. 
Pilot Study:  A double-blind placebo-controlled pilot study investigated whether the nutritional 
supplement, Muscle Armor, affects immune function.  Since inflammation is associated with loss of 
muscle during aging and wasting disease, it was hypothesized that Muscle Armor functions, in part, by 
exerting anti-inflammatory effects.  It was also hypothesized that the supplement would boost the 
response to vaccination, the gold standard measure of immune function.  Subjects were randomized to 
consume Muscle Armor (N=6) or placebo (N=6) for 4-weeks.  The tetanus, diphtheria, and pertussis 
(TDAP) vaccine was administered two weeks after product consumption began.  Plasma was collected 
prior to the start of consumption, immediately prior to vaccination, and 1- and 2-weeks post-vaccination.  
Muscle tissue was obtained before and after consumption of supplement or placebo though analyses of 
these specimens are not yet complete.  Plasma C-reactive protein (CRP), tumor necrosis factor-α 
(TNFα), and interleukin-6 (IL6) were measured as systemic markers of inflammation.   
Results and Conclusions for Inflammation:  The results for CRP are shown in Figure 7.  After 4-
weeks of intervention, CRP levels decreased in the supplement group but not in the placebo group 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
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(mean ± SEM:  -1.37 ± 1.06 versus 0.42 ± 0.50 mg/L).  Based on analysis of log-transformed data, the 
difference between groups was not significant statistically (P=0.16).  Although the sample size was 
relatively small (N=12), these data provide a reasonable estimate of the expected effect size which was 
calculated to be 0.85 standard deviations.  This effect size is similar to the estimated effect size given 
for muscle macrophage content under Aim 1.  Thus, the power for Aim 1 has been updated to include 
this new calculation.  The proposed sample size (N=50 with 20% dropout) gives 80% power to detect 
an effect size of 0.85 or greater at a 5% significance level.  The results for TNFα (not shown) also 
suggested that the supplement functions, in part, by decreasing systemic inflammation though a 
change in IL6 due to the supplement was not detected.       
Results and Conclusions for Vaccination:  The results for the antibody response to the pertussis 
antigen are shown in Figure 8.  Median levels of antibody increased in both groups after vaccination.  
The increase from pre-vaccination was greater in the supplement than the placebo group at both 1-
week (37.8 v. 19.6) and 2-weeks (131.0 v. 70.7) post-vaccination.  Based on a Mann-Whitney test, the 
difference between groups in the 2-week change in pertussis antibody titers was not statistically 
significant (P=0.13).  Thus, it is unknown whether the proposed sample size (N=50 with 20% dropout) 
will allow detection of differences between supplement and placebo groups though the nonparametric 
test result based on only N=12 is suggestive that it may.  Regardless, the measure remains valuable to 
the study because it is a good general measure of immune function that is quite variable for older 
adults.  This variability is amenable to planned within group analyses to determine the correlation 
between immune function and muscle adaptation to training and detraining.  This issue was previously 
addressed in the pitfalls section of the proposal is reiterated here due to its importance.  The aims were 
purposefully designed to be independent so that conclusions can still be drawn for the relationships 
between immune function and training outcomes as well as supplementation and training outcomes 
even if immune function is not altered by supplementation.     
 
 
 
Figure 7.  Change in CRP levels after 4-weeks of 
consumption of Muscle Armor or placebo.  Mean 
and standard error of change are shown. 
 
Figure 8.  Effects of Muscle Armor or placebo 
consumption on median response to the pertussis 
vaccine at 1- and 2-weeks post-vaccination. 
 
 
 
 
 
 

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Richard Dennis, PhD 
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RESEARCH DESIGN & METHODS 
Study Summary (Figure 9):  Veterans (age 60-80 yrs, N=50) will participate in a randomized double-
blind placebo-controlled trial.  The trial will evaluate the effects of a nutritional supplement (Muscle 
Armor) on immune function, muscle adaptation to resistance training, and retention of exercise benefits 
after training is halted.  Individual participation will last approximately one year (≥ 47 weeks).  This time 
will include periods for screening and introduction to exercise, supplementation prior to training (2 
weeks), supplementation plus supervised resistance training (36 sessions), and supplementation during 
post-training follow-up (26 weeks).  Subjects will undergo four muscle biopsies and blood draws to 
measure the effects of supplementation on the immune response to a bout of exercise.  A fifth biopsy 
will be collected to measure cellular adaptations in muscle to resistance training.  Five additional blood 
draws will occur to assess eligibility, safety, markers of inflammation, and the response to vaccination 
for tetanus, diphtheria, and pertussis (TDAP).  Strength will be measured bi-weekly, mass and function 
will be measured at baseline, and after the training and post-training/detraining periods.   
Figure 9:  Approximate timeline of subject participation and summary of trial protocol. 
 
X/X denotes assessment of response to a single bout of exercise by collecting tissue before and after 
exercise for blood (1 hour) and muscle (72 hours).  The above time-frame is optimal though only an 
approximation because of scheduling issues related to federal holidays, subject availability, etc.  
 
Key procedures to be completed by veterans (age 60-80, N=50): 
o Pre-screening with required waivers and permissions 
o Enrollment screening after written consent and HIPAA 
o Approximately 50-60 research visits over ~1-year 
o Data collection from CPRS problems and medication lists 
o Orange-flavored supplement or placebo consumption 
mixed in water twice daily (~42 weeks) 
o High-Intensity Resistance Exercise (38x over >12 weeks) 
o Post-training follow-up (~26 weeks) 
o Blood draw (9x) 
o Muscle biopsy (5x) 
o Gait and balance testing (4x) 
o TDAP Vaccination (1x) 
o Thigh CT Scan (3x) 
o Compensation (~$1500):  $20 per visit plus bonuses 

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HUMAN SUBJECT ASPECTS OF THE STUDY 
1. Recruitment (Target Population):  Veterans of age 60-80 years will be recruited.   
a. Accruals Requested:  brief pre-screening with required waivers (N=8000), sign consent and 
undergo screening (N=200), participate in study (N=100), complete all aspects of study 
considering possible dropout (N=40 to 50).  These number will encompass all approved 
recruitment methods. 
b. Permission is requested for N=50 to complete the exercise and supplementation protocol 
after consent and screening.  If withdrawal or dropout occurs, no additional data will be 
collected but specimens or data already collected from the subjects will be used unless the 
subject requests otherwise.   
c. Recruitment will not target vulnerable populations.  Pregnant women and fetuses, prisoners, 
and children are excluded from research due to both VA policy and by the purpose of the 
study.  Should a subject become incarcerated, he or she will be withdrawn from the study by 
the investigator.  If the incarceration is only temporary, does not interfere with subject’s 
ability to participate, and the event is discovered by the investigators after the fact, the IRB 
will be consulted as to whether the subject must be withdrawn. 
d. Other vulnerable populations (economically and educationally disadvantaged persons, 
employees and students, and homeless persons) will not be targeted for recruitment but are 
not specifically excluded.  An older veteran may respond to recruitment who by coincidence 
also happens to be a member of one of these other vulnerable populations.  These 
individuals will undergo the same informed consent process and will be afforded the same 
research protections as any other participant. 
e. Individuals with diminished cognitive capacity may possibly respond to the recruitment since 
the study is targeting older (60-80 yrs) adults.  However, individuals lacking the cognitive 
capacity to consent, as judged by a study physician, will be excluded either during the pre-
screening or screening phase.  The PI will exclude any individuals who appear unable to 
understand the consent process. 
f. Male and female veterans are eligible. 
g. Minorities are expected to respond to recruitment to the extent of their representation in the 
local VA population.   
2. Recruitment (Advertising):  Recruitment will occur from flyers (e.g. post-cards or electronic) 
placed within the CAVHS LR and NLR campuses and by other methods described below.  All 
advertising materials will have been approved by the IRB and the CAVHS Public Affairs office.  
a. Newspaper advertisement may be used but not necessarily due to the high cost of the local 
newspapers (Democrat Gazette) including military specific publications such as the Combat 
Airlifter and North Pulaski Leader.  Aging Monthly may also be used.   
b. Word-of-mouth may also be used as a recruitment method based on recruit referral from 
physicians, our subjects, and other individuals.  
c. Flyers and post-cards may be posted within the community at military specific locations such 
as the American Legion and the VFW.  Non-military specific locations in the community, 
particularly areas frequented by older adults, may also be used. 
d. These recruitment methods most always lead to the initial contact with the veteran being 
when a recruit calls the study.  However, it is possible that the contact could also be in 

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person.  Regardless, “cold-contacts” will not be used.  Contact will not involve procedures or 
data elements except as permitted by the approved protocol. 
e. Two new methods of recruitment are requested with the 07-25-2016 modification.  The 
study will recruit by mailings based on search of patient records and also using a staffed 
information table in concourse areas of the CAVHS hospitals. 
i. Need:  The study needs to mail an advertisement letter (included in submission) in 
order to recruit participants.  This recruitment method is necessary since the use of 
paper flyers has been banned on the CAVHS campuses.  The study is currently 
using post-cards displayed around campus but they are not effective compared to 
flyers.  The electronic message boards are supposedly available but the study has 
been unsuccessful in posting on the boards despite requesting access for the past 
three months.  The study also needs to place staff at an information table in 
concourse areas (e.g. off to the side in concourse outside hallway to primary care).       
ii. Justification:  From 08/2014 to 04/2016 the study received an average of 21 phone 
calls per month from interested Veterans based on flyer postings and placement of 
four newspaper ads at a cost of ~$1000 for three days each.  Since the 04/2016 
banning of flyers, the call rate has averaged only 10 participants per month though 
this number was helped by the placement of one newspaper ad.  More importantly, 
the use of post cards rather than flyers has skewed the recruitment response 
towards less healthy individuals.  This claim is based on the success rate of 
individuals making it past the initial eligibility screening by phone which changed from 
more than four per month to less than one per month.  The presumed reason is that 
flyers are the advertisement method that Veterans are familiar with and accept 
whereas post-cards are only likely to be noticed by patients who spend much more 
time at CAVHS.  
iii. Method 1:  The study will mail an advertisement letter (opt-in) to individuals 
identified through screening primary care appointment lists available in CPRS.  
These lists are available to anyone with CPRS access.  The main benefit of this 
method is efficiency since these Veterans should have an up-to-date health 
assessment from the primary care visit.  A checklist (included with submission) will 
be used to determine if individuals meet basic general eligibility criteria.  Records will 
not be accessed if marked “sensitive” or if the person does not meet the age 
requirement.  If all criteria aside from the latest PC visit are met, then the person’s 
name, last four of SSN, and address will be recorded on a checklist.  Then later (e.g. 
next day) the primary care assessment will be reviewed and if no concerns exist, 
then an advertisement letter will be mailed.  Individuals will be contacted and further 
recruited only if they respond to the letter.  Those who respond will continue 
recruitment by the already approved method.  Non-responders will be placed on the 
recruitment/enrollment log (name only) so that they are not contacted repeatedly.  In 
order to perform this process waivers of HIPAA and informed consent (see 
Supplement L) are requested.   
iv. Extension of Method 1:  The package 28 modification requests permission to 
extend the CPRS appointment list screening of recruits to all CAVHS clinics not just 
primary care.  Permission is also requested to extend the CPRS screening and the 
mailing of opt-in advertisement letters to past participants in our studies 204327 and 
391351 (Waiver of HIPAA and Consent has been requested to access name and 
last four from those study charts).  The already approved process and protections for 

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screening and mailing of the opt-in letters of invitation also applies to these 
extensions. 
v. Method 2:  The study will also place staff members at information tables available to 
Veterans.  The most likely area is in the concourse between the elevators and the 
hall to primary care in NLR (similar to the Million Veterans Program).  Staff will offer 
verbal explanation or an already approved post-card, or conduct the already 
approved initial contact discussion (see pre-screening process below) if the Veteran 
is interested.  The IRB can be confident that this face-to-face encounter will not 
include a coercive “sales pitch” as drop out severely hurts enrollment.  Participants 
join the study due to personal interests in exercise and nutritional supplementation 
and the willingness to attend approximately 50 visits over the course of a year.  
Stated another way, the study never attempts to “talk someone into” this substantial 
commitment. 
vi. Additional Protections:  Any Veteran information collected by the processes 
described above will be protected using the same VA confidentiality and privacy and 
protocol information security mandates already adhered to by the study.  Every effort 
will be made to ensure that these processes are seen as non-intrusive and 
respectful of privacy from Veterans or the process will be modified if needed.  
Appointment lists will not be printed.  As with all other study records, paper 
documents are stored behind a least two locks in the GRECC in the office of the PI 
or temporarily with the coordinator and electronic records are stored in the PI’s 
folders on the GRECC server.              
3. Pre-Screening:  Pre-screening for eligibility will be performed when a recruit responds to 
recruitment, is interested in learning more about the study, and is interested in being considered 
for participation.  A request for waivers of HIPAA and written consent is limited to this specific 
minimal risk activity.  Recruits who appear eligible will come to the NLR VA for a research visit to 
continue the informed consent process, including written consent, HIPAA authorization and further 
screening for eligibility.  Thus, the main purpose of the pre-screening is to minimize the chance of 
a recruit who does not meet even the basic eligibility criteria from unnecessarily attending a 
research visit.   
a. Verbal Informed Consent:  Verbal informed consent will be obtained by telephone to 
conduct the pre-screening.  Recruits must answer affirmatively to having an interest in being 
considered for the study for the pre-screening to continue.  Recruits are told that they may 
refuse to answer any question.  This activity presents minimal risk to the recruit and all 
information will be protected as confidential.  Justifications are included in the IRB-required 
supplement included with this submission.  
b. Waiver of HIPAA:  Permission is requested to for a waiver of HIPPA in order to collect pre-
screening information from the recruit and from CPRS.  Pre-screening information will be 
documented on the “Initial Contact Form” and “Medical History and Physical Exam Form” as 
applicable.  Justifications are included in the IRB-required supplement included with this 
submission. 
c. Initial Contact and Pre-Screening:  Documentation of the pre-screening (partial list of 
eligibility criteria) will begin on the “Initial Contact Form”.  The form contains a scripted 
explanation of the study to be used by a member of the study team who has been delegated 
this responsibility and trained by the PI.  The brief explanation describes study procedures, 
benefits, and risks.  The form contains the last 4 digits of the SSN which will be used to 
access the recruit’s CPRS record if verbal consent to pre-screening is given.  

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i. If the recruit gives permission for CPRS pre-screening, then the CPRS Problems List 
for the recruit will be reviewed by the study physician in order to assess for exclusion 
criteria.  This review will be documented on the “Medical History and Physical Exam 
Form”.  This screen of the CPRS Problems List includes among the criteria, a check 
for evidence for lack of cognitive capacity to consent for the study. 
ii. Recruits who appear eligible will be contacted to schedule a research visit in which 
written consent will be obtained and the screening process will be continued by the 
study physician.  It is not a necessity but recruits are asked to bring the following 
information to their visit:  list of current medications; names and telephone numbers 
of two emergency contacts; name and number of personal physician.   
iii. Consent to the CPRS pre-screening process is not a requirement to be considered 
for the study.  Individuals who wish to be pre-screened after written consent will be 
allowed to do so during a research visit. 
iv. Obviously CPRS pre-screening does not apply to recruits who have not previously 
received VA healthcare.  These recruits will be informed that a DD214 is necessary 
and that a VA medical record must be created for them prior to written consent. 
4. Informed Consent Process and Written Consent:  Recruits passing the pre-screening will 
attend a research visit to consent the consent process, provide written consent and continue 
eligibility screening.  The goal of the informed consent process is to ensure complete disclosure 
and voluntary participation for the subjects.   
a. Informed Consent Process:  Informed consent will be an ongoing process that begins 
when the recruit first learns of the study and will continue as an ongoing exchange of 
information between the research team and the subjects throughout the course of the study. 
b. Written Informed Consent:  Written informed consent will be obtained from recruits by the 
PI or a trained member of the study team who has been delegated this responsibility.  The 
current IRB-approved and stamped version of VA Form 10-1086 will be used.  Written 
consent will be obtained prior to the continuation of the screening for eligibility.   
i. The process will be conducted in the English language. 
ii. The informed consent form will have been mailed to the participant after the pre-
screening if practical.  If mailing the consent is not practical such as if the study 
appointment is the next day, then the recruit will be given as much time as needed at 
the research visit to read and understand the consent form.   
iii. The elements of the consent form will be discussed with the recruit in an area where 
privacy can be maintained.  The recruit will be told that participation is completely 
voluntary and that refusal to participate will not influence his healthcare or have 
negative consequence. 
iv. Recruits will be allowed to ask questions and express concerns.  If the recruit feels 
that he or she has not had sufficient opportunity to consider whether or not to 
participate in the research, he or she will be allowed to leave and return at a later 
date.  Alteration of the appointment schedule to ensure subject consent, safety, 
convenience, etc will not be considered a protocol deviation. 
v. If during the consent process, the person obtaining consent believes that the recruit 
does not understand the procedures and risks of the study or the consequences of 
not participating, then written consent will not be obtained and the person will be 
excluded from the study.  The process or the reason for not obtaining consent will be 
documented in the consent process note.   

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vi. Subject will be given a copy of the signed consent form and the original will be kept 
in the subject file. 
vii. Consent will also be documented in a consent process note placed in CPRS and the 
subject file within 24 hours of obtaining consent.  All subjects participating in the 
greater than minimal risk aspects the study will have their CPRS record flagged with 
a Category II patient record flag.  The flag will be removed once participation has 
completed or the subject has been withdrawn and 30+ day monitoring is complete.   
viii. The PI will sign the consent document and the document will be hand delivered for 
scanning within the IRB required time frame of the subject signature.  The 10-9012s, 
HIPAA, and scanning request form will also be included. 
5. Screening for Eligibility:  Subjects who provide written informed consent will undergo a 
screening for eligibility by the study physician using the Medical History and Physical Exam form.  
The inclusion and exclusion criteria are based on the study design and health suitability to 
undergo exercise training, supplementation, blood draws, biopsy, CT scan, and vaccination.  If 
exclusion criteria are temporary, recruits may participate in the study (or specific procedure 
related to criteria) once condition is no longer exclusionary.  The criteria  and method for collection 
of this information are listed: 
a. Inclusion Criteria:  information collected from subject self-report 
(1) 
Veteran 
(2) 
Age 60-80 years  
(3) 
Body Mass Index of 18.5 - 29.9 kg/m2 
b. Exclusion Criteria: information collected from subject self-report  
(1) 
Currently participating in any other research study involving an intervention 
(2) 
Smokes tobacco products (subject word to halt smoking is also sufficient) 
(3) 
Tetanus or TDAP vaccine in previous two years 
(4) 
Allergic to vaccination 
(5) 
Seizure in past 3 months 
(6) 
Guillain-Barre Syndrome in past 3 months 
(7) 
Takes the medications heparin, plavix / clopidigrel, or coumadin / warfarin 
(8) 
Allergic to lidocaine 
(9) 
Significant problem with fainting 
(10) Problems walking or exercising with both legs   
(11) Participated in a weight-lifting program targeting the thighs in last 3 months 
(12) Pains, tightness or pressure in chest during physical activity such as walking, climbing 
stairs, household chores, or similar activities 
c. Absolute Exclusion Criteria:  information collected from CPRS review and/or subject self-
report.  The medical history will include a discussion between the subject and the study 
physician of the exclusion criteria that may have been reviewed during the pre-screening of the 
CPRS Problem List.  The physician will assess the available information and document whether 
or not the subject is excluded. 

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(1) 
Enrolled in another interventional study (CPRS Category II Flag) 
(2) 
Metastatic cancer or undergoing chemotherapy 
(3) 
Cerebral aneurysm or intracranial bleed in past year 
(4) 
End-stage congestive heart failure (NYHA Stage IV) 
(5) 
Unstable abdominal or thoracic aortic aneurysm (>4cm) 
(6) 
Renal disease requiring dialysis 
(7) 
Allergic to lidocaine or vaccination 
(8) 
Acute retinal hemorrhage or ophthalmologic surgery in past 3 months 
(9) 
Bone fractures in the pelvis, legs, or feet in the last 3 months 
(10) Hernia that causes pain during physical activity 
(11) Myocardial infarction or cardiac surgery in past 3 months 
(12) Pulmonary embolism or deep venous thrombosis in past 3 months  
(13) Proliferative diabetic retinopathy or severe nonproliferative retinopathy 
(14) Active suicidality or suicidal ideation 
(15) Systemic bacterial infection 
(16) Taking heparin, plavix / clopidigrel, or coumadin / warfarin 
(17) Taking ASA (in any form) and unable/unwilling to discontinue at least 10 days prior to 
muscle biopsy 
(18) Unwilling to halt concurrent use of amino acid or protein supplements 
(19) Unwilling to halt new use of nutritional supplements 
(20) Unwilling to maintain current normal diet 
(21) Guillain-Barre Syndrome in past 3 months 
(22) Tetanus vaccination in past 2 years 
(23) History of allergic reaction to vaccination 
(24) Encephalopathy in past 7 days 
(25) Active oral or genital herpes 
(26) Current use of appetite stimulants 
(27) Significant history of seizures or seizures in past 3 months 
(28) Current treatment for mania or bipolar disorder or taking lithium. 
d. Potential Exclusion Criteria:  evidence for potential exclusion criteria will be collected 
from CPRS review and/or subject self-report.  *Certain issues may also be assessed by the 
physical exam.   
(1) 
Diagnosis of a cognitive deficit 
(2) 
Untreated severe aortic stenosis:  if there is evidence in CPRS Problem List or subject 
self-report of untreated aortic stenosis, then physician judgment for inclusion must be 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
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based on an available echocardiogram, or evidence a valve area of < 1cm2, or a history in 
the past 3 months of syncope, heart failure, or angina. 
(3) 
*Uncontrolled diabetes mellitus (HbA1C>10) 
(4) 
*Uncontrolled hypertension or hypotension (>160/100, <100 systolic) 
(5) 
*Uncontrolled malignant cardiac arrhythmia (sustained ventricular tachycardia > 3 beats, 
complete heart block, atrial flutter, symptomatic bradycardia) 
(6) 
Unstable angina (at rest or increased pattern in past month) 
(7) 
Allergic to latex or tape 
(8) 
Bleeding or clotting disorders, family history of bleeding disorders, problems with abnormal 
bleeding  
(9) 
Taking any non-ASA NSAID and unable or unwilling to discontinue use for 3 days prior to 
the muscle biopsy procedure 
(10) Taking Fish Oil, Gingko, Garlic, Saw Palmetto, Turmeric, or Vitamin E and unable or 
unwilling to discontinue use for 10 days prior to the muscle biopsy procedure.  Multivitamin 
containing vitamin E is acceptable and does not need to be halted. 
(11) Significant history of fainting 
(12) Significant problems with chronic pain 
(13) Uncontrolled asthma or allergies 
(14) Taking lactulose, nitrates plus hypertension medications or Viagra  
(15) Liver cirrhosis or other severe liver disease 
(16) History of peripheral artery disease 
(17) Steroid or androgen use in past 3 months that physician feels may confound study 
(18) Other physician judgment 
e. Other Exclusion Criteria:  information collected during the physical exam by the study 
physician or qualified designee.  Blood will be drawn (less than or equal to 60ml or 2 ounces) 
for lab analyses by the hospital clinical lab at study entry, after completion of the exercise 
training program, and upon completion of the study for tests related to eligibility, safety, and 
research data.   
(1) 
Blood Pressure greater than 160/100 or <100 systolic. 
(2) 
HbA1C>10 
(3) 
CBC or PT/PTT/INR results that the study physician determines indicate a risk for 
excessive bleeding. 
(4) 
Clinical labs performed for safety monitoring including AST, ALT, creatinine, and BUN 
(5) 
Other tests that performed to measure immune function include HbA1C, CBC, CRP, ESR, 
cholesterol HDL and LDL, and blood differential.  
f. Exclusion Criteria Based on Other Physician Judgments:  older veteran subjects often 
possess numerous comorbidities that result in a complex and unique set of health issues.  Thus, 
in addition to the specific exclusion criteria named above, the physician must also use a 
considerable amount of judgment in determining whether a recruit should be excluded even 
though the recruit meets the named inclusion criteria and does not meet the named exclusion 

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criteria.  The physician also makes such judgments to determine if a subject should be 
withdrawn once participation has begun.  The physician may obtain any additional unforeseen 
medical information that may be needed at enrollment or during participation.  As explained in 
the consent and HIPAA documents, this information may be collected through CPRS, additional 
examinations or tests, or by consultation with the subject’s healthcare providers from inside or 
outside the VA.  As appropriate, the physician will document and use this information to judge 
whether the subject can safely participate, should be excluded or withdrawn, or should be 
referred for treatment. 
6. Scheduling:  A scheduling calendar will be used by the study staff and study visit scheduling cards 
will be given to the subjects to help ensure compliance with study visits and procedures. 
a. A subject scheduling calendar will be maintained in the form of an Excel spreadsheet.  The 
calendar is needed in order for the study team to coordinate study procedures and data 
collection particularly when multiple subjects are scheduled for any one day.  Thus, the calendar 
will improve study efficiency, subject safety, and care.  The calendar will contain dates, subject 
ID #, and procedures for each visit.  It will be stored in the PI’s secure folder containing 
electronic logs.   
b. The study will also give patients visit cards that will include subject name, visit date and time for 
up to three visits, procedures, and estimated time required.  The calendar and cards will help 
keep the study team and subjects on track with the study requirements for each visit. 
c. The scheduling cards will typically be hand given to subjects at an appointment when their next 
appointment time and date is already known.  However, scheduling cards may also be mailed, 
as needed, for instances when an appointment must be scheduled over the phone and the 
subject desires a written reminder in addition to the reminder telephone calls that are already 
commonly used.  Obviously a card would not be mailed, for example, if a subject is contacted by 
phone and states that a card is not needed or if the appointment is scheduled for so soon as to 
not allow time for mail to be received.     
7. Payment of Subjects:  This research is not integrated or related to patient care.  Subjects will be 
compensated for their time and inconvenience and as incentive to participate in compliance with the 
requirements of the study as explained in informed consent. 
a. Payments will be made in the form of Kroger gift cards. 
b. Participation involves approximately 50-60 research visits over the course of approximately one 
year.  Subjects will receive $20 for each visit attended.  However, approximately 10 of the visits 
(e.g. once every four weeks) will require that the subject return a dosing calendar and the 
canisters containing any unused portion of supplement or placebo.  This process is required to 
monitor subject compliance with product consumption.  At these visits, subjects will receive the 
$20 if the dosing calendar and canisters are returned.  If a subject forgets to bring these items, 
they can be returned at the next visit and the payment will then be given.  If these items are 
never returned, the subject will not receive that one payment.    
c. The two visits in which the subject undergoes blood draw, biopsy, exercise, and blood draw 
require approximately 4 hours.  For each of these two visits the subject will receive an additional 
$20 gift card (i.e. $40 for the visit).  

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d. Subjects will be given two completion bonuses.  Subjects will receive $100 after completion of 
the resistance training program and $300 after completion of the entire study.  The total amount 
earned for completion of all study visits will total approximately $1,500.  
e. The compensation is normal compared to similar studies conducted at other institutions.  The 
amount is not considered coercive considering that subjects will have more than 50 visits over a 
one year span that require a high level of conscientious active participation (exercise training 
and accurate supplement/placebo consumption, multiple biopsies and blood draws). 
f. The consent form will inform the subject of that the VA may report participation payments as 
income to the IRS. 
g. Finder’s fees and recruitment bonuses will not paid to VA or study staff.  If a subject refers 
another veteran to the study who signs consent to participate, then a $20 gratuity will be given 
to the referring subject. 
8. Randomization:  After medical clearance to participate, subjects will be randomized to either of two 
parallel groups, treatment with Muscle Armor or placebo.  The double-blind randomization will be 
performed by a research pharmacist.  Assignments will be unknown to investigators and subjects.  
The pharmacist will review all study material prior to approval, and can break the blind if needed for 
safety purposes, but is otherwise uninvolved in the study.  The pharmacist will dispense the 
products in sealed and blinded packaging.  The study will appoint a study monitor (specific un-
blinded member of the study team) to review and be involved in pharmacy and subject compliance.  
This person will not be involved in subject assessments or specimen analysis. 
9. Supplementation:  Muscle Armor was chosen from a scientific standpoint because of the ability of 
its ingredients to modulate muscle health and immune function.  Muscle Armor is a good choice for 
practical purposes because it’s ingredients are safe to consume [46;47] and it is available to 
individuals by retail. 
a. Subjects in the supplement group will consume orange-flavored Muscle Armor according to the 
manufacturer’s directions:  one serving (approximately 30g, i.e. one scoop provided with the 
product by its manufacturer), twice daily (optimally morning and evening), mixed with 12 ounces 
(oz) of water.  Subjects in the placebo group will similarly consume one serving (approximately 
13g, one scoop provided to pharmacy by the study) of orange-flavored Kool-Aid mixed with 12 
ounces of water twice daily. 
b. Quality control weighing of single doses of supplement and placebo indicates that exact dose 
will vary based on whether a level, sub-level, or heaping scoop is used and this is acceptable.  
Dose range for the supplement appears to be 30-35 grams and dose range for the placebo 
appears to be 11-15 grams.  This is less than the actual single serving size listed on the 
instructions for Kool-Aid but is deemed acceptable due the sugar content of the recommended 
serving size since diabetics will likely be enrolled and older adults may dislike the taste due to it 
being too sweet.   
c. Supplementation will typically begin the day of the second muscle biopsy (typically Visit 5) and 
continue for the remainder of the participant’s study involvement (optimally approximately 42 
weeks).  The total dosing time will likely be longer than the optimal time because scheduling of 
the exercise training and other appointments must be performed based on VA holidays, patient 
and physician availability, etc.  And specific starting date or intermittent dosing lengths of time 
can be altered without protocol deviation in order to maintain proper spacing of the more time 
dependent clinical procedures, e.g. blood draw, biopsy, and vaccination.        
d. Compliance will be monitored using subject self-report and both the number of doses consumed 
and weight of product consumed.  Each canister will contain 14 doses (7 day supply) though 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
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this number is approximate due to the inexact measurement of one scoop.  Canisters will 
contain 421 grams of supplement or 188 grams of placebo.  Subjects will return empty 
containers.  Containers will only be weighed if unused supplement or placebo is returned.  Self-
report will be based on completion and return of the dosing calendar.  This information will be 
collected and reviewed by a study monitor, i.e. member of the study team who is not blinded.  
The monitor is planned to review dosing and consumption for compliance with the subject 
approximately every four weeks by phone or in person or more often as needed.  Subject diet 
data will not be collected but to be involved subjects must agree to maintain their normal diet 
during participation. 
e. Subjects will be given a gym bag to transport their canisters of supplement/placebo and 
consumption calendar between home and study visits.  They will also be given two drink mixer 
bottles of the type commonly used to mix powder shakes.  These products contain the GRECC 
name and phone number of the study coordinator.  Subjects will be allowed to keep the bag and 
bottles upon completion of the study.  The approximate value of the products per subject is 
~$28.  These gifts are justified due to making it easier for the subject to transport materials to 
and from study visits which should increase subject compliance with dosing and monitoring 
requirements. 
f. 06/12/2017 Modification.  The study has learned that Abbott Nutrition is no longer manufacturing 
Muscle Armor and will only sell a similar product, Juven, in the future.  The study will switch to 
using Juven with approval of this modification though remaining stores of Muscle Armor will be 
used until expiration date.  The study had originally planned to use Juven but did not use Juven 
after learning that it is only available in single serve packets which would make the blinding 
process laborious.  Use of Juven will require the study team member who packs the placebo in 
the pharmacy to pack the supplement Juven in similar fashion, i.e. 14 doses (packets) will be 
emptied into the usual blinded supplement canisters.  Other pertinent details of the switch 
include the following: 
i. Juven and Muscle Armor are essentially the same product but were being 
marketed by the company under two different names.  They taste, smell, and 
look identical.  Thus, the blind will not be in jeopardy due to the switch. 
ii. The key ingredients of interest (Arginine, Glutamine, Methylbutyrate) are present 
in the same amounts in Juven and Muscle Armor.  Juven lacks the 3g Taurine 
that is in Muscle Armor and Juven contains phenylalanine, and FD&C Yellow #6 
instead of Red #40, and aspartame instead of sucralose.   
iii. The product identity and ingredients were not mentioned in the consent form or 
other information given to subjects.  
iv. Since Taurine is missing in Juven, the dose size is slightly smaller than Muscle 
Armor (24g versus 30 g).  However, Juven was measured in the same scoop 
size used for Muscle Armor and the difference was negligible. 
v. Switching to a slightly different product during the study is not optimal but is 
necessary.  The switch is not expected to affect the results.  However, the study 
will track when the switch was made for each subject to determine during 
statistical analysis if a bias was introduced.     
g. Benefit:  The subjects may or may not have improved muscle health and immune function due 
to the supplement.  The supplement may improve the response to the vaccine.  However, it is 
unknown whether this improvement will convey increased protection from disease.   

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h. Risk:  There is little evidence from controlled studies of risk associated with the ingredients of 
the supplement.  However, adverse events have occurred in which the relation to underlying 
disease or the supplement was not discerned.  Thus, this study will take a conservative 
approach and consider that the ingredients may exacerbate the following conditions:  allergies 
or asthma, herpes, and low blood pressure. The supplement may cause side effects such as 
abdominal pain, bloating, diarrhea, gout, blood abnormalities, sensitivity similar to monosodium 
glutamate (MSG; headache, flushing, tingling, weakness).  Caution should be used when taking 
the supplement and lactulose.  The supplement should not be taken by individuals undergoing 
chemotherapy, or those having a recent heart attack, liver disease, seizures, mania or bipolar 
disorder and taking lithium.   
i. 
 
Table 2.  *Comparison of nutrient information between Muscle Armor supplement and Kool-Aid 
placebo control for one serving mixed with 12 oz water.  Juven ingredients listed in parentheses. 
MUSCLE ARMOR 
PER SERVING 
KOOL-AID 
PER SERVING 
AMINO ACIDS 
 
 
AMINO ACIDS 
 
 
L-ARGININE 
7 (7) 
g 
L-ARGININE 
0 
G 
L-GLUTAMINE 
7 (7) 
g 
L-GLUTAMINE 
0 
G 
TAURINE 
3 (0) 
g 
TAURING 
0 
g 
CARBOHYDRATE 
10 (8) 
g 
CARBOHYDRATE 
16 
g 
SUGARS 
5 (2) 
g 
SUGARS 
16 
g 
MINERALS 
 
 
MINERALS 
 
 
CALCIUM 
200 (200) 
mg 
CALCIUM 
0 
mg 
Calcium β-hydroxy-β-
methylbutyrate 
1.5 (1.5) 
g 
Calcium β-hydroxy-β-
methylbutyrate 
0 
g 
VITAMIN C 
0 (0) 
mg 
VITAMIN C 
9 
mg 
ENERGY 
110 (80) 
kcal 
ENERGY 
60 
kcal 
 
*Juven supplement has the same composition as Muscle Armor except it lacks Taurine. 
j. 
Minimization of risks:  The risks of participation will be minimized by cautious consideration of 
the potential adverse effects of Muscle Armor by the study physician during enrollment and 
monitoring.  Individuals who should not take the supplement will be excluded.  Subjects will be 
informed of the potential side-effects and advised to report any symptoms.  Symptoms related to 
the supplement and other study procedures will be monitored for, as applicable, at each study 
appointment and by attempted telephone contacts weekly.  Safety monitoring will also include 
blood draws for assessment of liver and kidney function at baseline, after completion of the 
training program and at the end of the study. 
9. Vaccination:  The International Society of Exercise and Immunology considers response to 
vaccination the gold-standard for assessment of immune function in the context of exercise [38].  
Multiple vaccine options were considered for use in the proposed study.  Vaccination with antigen 
more likely to be naïve to subjects is preferable.  However, the risk to benefit ratio of using the 
keyhole limpet protein or the herpes zoster live-attenuated virus was considered unacceptable.  The 
influenza vaccine was considered since the epitope may change yearly, but this option could limit 
recruitment outside of flu season by availability of the vaccine and during flu season by availability 
of unvaccinated veterans.  The tetanus, diphtheria, and pertussis (TDAP) vaccine has the 
advantages of benefit to the subject, low risk, low cost, and available test kits to measure response.   
a. Procedure:  The vaccine will be administered by a study physician to each subject after 2-
weeks of supplementation.  Blood will be collected at baseline and 1 and 2-weeks post-
immunization to determine the effects of the supplement on the humoral response.  Two time-
points were chosen in case the response is delayed in older adults relative to the response in 

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younger adults [48].  If needed to accommodate scheduling, the supplementation period may be 
extended.  The vaccination and second biopsy may occur up to 5 days late and the two post-
vaccination blood draws may be delayed by one day if necessary.   
For each study procedure a qualified study staff member will perform the procedure, i.e. a study 
physician will administer the vaccine and the study coordinator will draw blood.  However, non-
study staff such as the GRECC nurse or a CAVHS phlebotomist may also be utilized as 
needed, without being added as study staff, if the activity falls within their CAVHS job duties.      
b. Benefit:  The TDAP vaccine was chosen because of the direct benefit it will provide to older 
subjects [49].  Tetanus, for example, is uncommon in the United States, but 70% of cases 
involve older adults and individuals over 60 years of age experience greater than a 50% 
mortality rate [50;51].  However, problems with these diseases are avoidable because the 
vaccine is safe for use in older adults.   
c. Risk:  The most common problems in adults are mild or moderate:  pain at injection site (mild 2 
in 3, moderate 1 in 100); redness or swelling at site (mild 1 in 5, moderate 1 in 25, severe 3 in 
100); fever (mild 1 in 100, higher 1 in 250); headache (mild 3 in 10, moderate 1 in 300); 
tiredness (mild 1 in 4), and nausea, vomiting, diarrhea, or stomach ache (mild 1 in 10, moderate 
1 in 100).  Chills, body aches, sore joints, rash, or swollen glands are uncommon.  A severe 
allergic reaction could occur after any vaccine (rare, less than once in a million doses).  
Individuals who are allergic or have experienced unexplained problems (coma, seizures) after 
vaccination, or has had Guillian-Barre Syndrome should not be vaccinated.  
d. Minimization of risk:  The most common problems can be safely managed with symptomatic 
treatment.  Individuals who should not be vaccinated will be excluded.  The procedure will be 
postponed until recovery for subjects with mild illness or fever.  All subjects will be informed of 
the risks and advised to call the study physician or staff immediately should a severe reaction 
occur.   
10. Resistance Exercise Training Program:  The study will use a resistance exercise routine that we 
have previously used to elicit cellular and molecular responses in muscle associated with immune 
function and to improve muscle mass and strength of the thigh for older adults [2;3;40;52].   
a. Procedure:  The resistance exercise program will use three exercises to strengthen the thigh 
muscles including bilateral knee extension, knee curl, and leg press.  Exercises are completed 
on air-driven exercise equipment in a seated position.  Exercise sessions are always supervised 
by a study team member. 
b. Strength Testing:  Strength will be determined using the 1-repetition maximum test [52].  A 
brief warm-up period will include approximately 10 minutes of mild aerobic activity followed by 
approximately 6 repetitions of lifting warm up at for a resistance exercise at a “somewhat heavy” 
load.  The load is then gradually (~5-10%) increased such that the maximum capability for lifting 
is reached in approximately 10 total repetitions.  Subjects will rest 60 seconds between 
repetitions after the warm up.  Subjects rest five minutes prior to the next test completed in the 
order of press, curl, and extension.      
c. Unaccustomed Exercise Sessions (2x):  Prior to exercise training, the study will measure the 
response to single bouts of the training exercises before and after two weeks of nutritional 
supplementation.  The response to exercise will be measured using both muscle and blood.  
The pre-exercise time points will be prior to exercise, possibly same say or a different day 
based on scheduling and/or the judgment of the study physician.   The post-exercise time points 
for collection are 72 hrs for muscle and 1 hr for blood.  Our studies have previously shown that 
72 hrs is a better than 24 hours for measuring the immune variables of interest in muscle [52].  

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The 1 hr time point for blood collection is based on other studies that have determined the 
effects of exercise on systemic measures of immune function [11;53].   
d. Exercise Training Sessions (36x):  The training program plans to have subjects perform 
supervised exercise at the NLR VA for approximately an hour optimally 3 times per week on 
non-consecutive days.  The program consists of 36 exercise sessions that will optimally take 12-
weeks though the total time will likely be longer due to rescheduling based on the needs of the 
subject or the study team.   
e. The exercise load will be based on how much the subject is capable of lifting one time.  After 
strength testing, subjects will perform exercises at loads ranging from 60% to 80% (e.g. sets at 
60, 70, 75, and 80% of capability) of their measured capability to the best of their ability.  
Strength is retested at approximately every 6th session to account for strength gain in 
maintaining the work load.  On days of testing, the strength test replaces one of the four 
exercise sets.  Strength is also tested twice prior to training, at the final training bout, and twice 
during the post-training follow-up period.  
f. Training sessions consist of three sets of 10 repetitions for each exercise and a 4th set 
completed at voluntary failure to complete a repetition.  Subjects will rest approximately two 
minutes between each set and approximately 5 minutes between exercises. 
g. Exercise data will be recorded for each subject on the Exercise Data Form. 
h. Exceptions to the plan for each subject to complete 36 exercise training sessions may be 
necessary on a case-by-case basis at the discretion of the PI in order to ensure that the final 
muscle biopsy of the protocol occurs 72 hours (3 days) after the final exercise training session.  
Addition or subtraction of a limited number (optimally 1-2) of exercise sessions may be 
necessary to meet this time frame due to scheduling difficulties for the biopsy.  Scheduling 
obstacles include physician or subject availability or more commonly the fact that the final 
exercise session cannot fall on a Wednesday or Thursday unless plans are made to perform the 
biopsy on Saturday or Sunday which is not preferable.    Maintaining this time frame for biopsy 
is the priority as it has a greater chance of influencing the cellular measurements to be made 
using the biopsy than would a small difference in the total number of exercise sessions for 
influencing strength gain over the long training period. 
i. 
Benefit:  Regular resistance exercise is an effective method for improving many aspects of 
health and well-being (13).  Subjects will likely improve the size (CT scan), strength (strength 
testing), and function (see below) for their thigh muscles.  The assessments of muscle function 
include: 
(1) Berg Balance Scale (BBS):  BBS is used to detect balance impairments in older adults 
while the individual is static or performing various movements [53].  The test consists of 
14 tasks performed in standardized order and the results have been shown to have good 
inter-rater reliability of 0.98. 
(2) Six Minute Walk Test (6MWT):  The test is used to measure mobility performance in 
older adults and individuals with limitations [54].  The subjects will perform a supervised 
timed walk on a 200 foot track at a pace without encouragement and which allows 
speech without shortness of breath.   
(3) Gait Speed:  The test measures habitual walking speed [55].  The subjects will walk at 
their normal preferred pace for 14 meters though only the middle 10 meters are timed.  
The result is an average of three replicates.   

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(4) Timed Up and Go:  The test is indicative of balance and mobility by a measuring the time 
required for the subject to rise from a chair, walk three meters, turn around, walk back to 
the chair, and sit down [55]. 
(5) Functional assessment data will be collected at four times during the protocol:  pre-
training, post-training, during post-training follow-up, and at end of study.    
j. 
Risk:  In all but limited contraindicating circumstances (our exclusion criteria), the benefits of 
regular exercise exceed the risks.  Exercise of the proposed intensity has been conducted 
safely in even frail elderly and adults in 80-90 years of age (8, 18, 23).  However, serious events 
are possible: 
(1) Exercise may cause (estimated at 1 of 100 cases or 1:100) shortness of breath, chest 
pain, or dizziness; and ligament, tendon, or muscle sprain.   
(2) Heart problems are a rare but serious risk of strenuous exercise and may result in 
sudden death (1:18,000 adults per year).  However, even in patients undergoing heart 
rehabilitation, heart failure (1:100,000), heart attack (1:200,000), and death (1:750,000) 
are rare events relative to the number of hours spent exercising. 
k. Minimization of Risk:  the risks of exercise will be minimized for the subjects as follows:   
(1) Each subject will have undergone a thorough medical history and physical exam 
including an electrocardiogram (resting and during exercise stress) performed by the 
study physician prior to participation.  Details and physician clearance to participate are 
documented on the Medical History and Physical Exam Form. 
(2) Subjects will not be allowed to begin exercise if their blood pressure is greater than 
160/100.  After training, if blood pressure is elevated, the subject will remain at the 
GRECC until it returns to normal.  If it does not return to normal the subject will be 
evaluated by a study physician. 
(3) Training will be supervised at all times during exercise by a member of the study team 
who is certified in basic life support to ensure subject safety and compliance.  If the 
subject cannot complete the exercise with proper form then the work load will be 
decreased or the set will be halted.  The member of the study team will know the 
emergency response procedures and they will be posted on the wall of the exercise 
room. 
(4) The exercises will be performed using air-driven equipment in a seated position.  Thus, 
there is no risk of the subjects falling, dropping weights, or having weights fall on them.  
(5) Subjects will complete an introductory exercise session in order to learn proper exercise 
form while lifting relatively light loads. 
(6)  Each training session will be preceded by a warm-up period on a stationary bicycle, 
treadmill, and/or the exercise equipment.  Stretching will be performed as needed during 
and after the training session. 
 
l. 
Emergency Response:  If there is a problem or concern for continued exercise, the person 
supervising the exercise will immediately halt the exercise.  This emergency response also 
applies to any potential problem occurring on-site.  If the problem is a potential emergency, the 
hospital medical response team will be called immediately at 6-911.  This number is posted on 
the telephone in the exercise training room.  The medical response team will arrive within 
minutes.  During this time, basic life support will be used if warranted.  If there is a medical 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
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problem that is not an emergency, the study medical staff and/or the GRECC nurse will be 
contacted to come evaluate the symptoms and determine a course of action. 
(1) Modification to Emergency Response Plan 09-16-2016: 
(2) This greater than minimal risk protocol is modified to inform the IRB that the emergency 
response procedure for an on-site serious adverse event has changed but there is no 
significant change in risk to the subjects.  Beginning the evening of 09-15-2016, the NLR 
campus will deploy a "medical response team" (MRT) instead of a "code team" in 
response to emergencies. 
(3) In response to email notification of this change on 09-13-16, Dr. Dennis Sullivan 
convened a 90-minute meeting (09-14-16) of the physicians and principle investigators 
of the GRECC.  Dr. Sheila Sullivan obtained further clarification on the details of the 
change from the Quality Management Coordinator.  We also reviewed the medication list 
to be included in the Stat Pack meds of the MRT.  We concluded that the risk posed to 
subjects participating in the ongoing clinical trials at GRECC and specifically this 
protocol, are not increased based on the following details of the change and our 
preparedness measures.  
(4) This opinion that risk to participants has not changed is consistent with the conclusion of 
research findings indicating that the provision of pre-hospital ACLS after cardiac arrest 
did not change the survival rate to hospital discharge [54]. 
(5) Under the new guidelines for emergency response, the medical officer of the day (MOD) 
will continue to respond as team leader of the MRT.  MRT will provide BLS and other 
care determined by MOD.  The patient will be transferred to an acute care facility, if 
needed, via MEMS (determined by MOD), as is similar to the prior practice. 
(6) Crash carts, including the crash cart on our unit, will no longer house medications.  
However, Stat Pack medications will be carried to each emergency site by a MRT nurse.  
The medications included are the same as those previously supplied on the crash cart 
with the exception of anti-arrhythmics and atropine.  However, our unit will stock atropine 
on site along with the items necessary for administration of these medications (e.g. IV 
lines and normal saline) so long as unavailable from the MRT.   
(7) Additionally, the MRT will not have access to intubation though use of this procedure 
was previously discouraged by the code teams anyway.  The MRT will still have capacity 
to support patient respiration (Ambu Bag) until MEMS arrives, if necessary. 
(8) The likelihood of serious adverse events with the protocol are low though the 
possibilities of concern include:  acute myocardial infarction (MI) due to strenuous 
exercise, vasovagal response with syncope and possible bradycardia during muscle 
biopsy, and anaphylaxis due to lidocaine sensitivity during muscle biopsy.  Baby aspirin 
and nitroglycerin are available in the Stat Pack in case of chest pain or other symptoms 
suggestive of MI.  As stated above, atropine and the means of administration will be kept 
on-hand in case of bradycardia.  Benadryl is available in the Stat Pack in case of 
sensitivity to lidocaine though epinephrine is also available from the MRT for serious 
reactions.   

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11. Post-Training Follow-up Period:  After completion of the training program, subjects will remain in 
the study for at least 26 additional weeks so that the retention of muscle mass, strength, and 
function can be examined relative to continued daily consumption of supplement or placebo.  
Subjects will be required to refrain from strength training of the thighs (i.e. weight lifting, body weight 
exercise, elastic bands) during this period.  It is acceptable for subjects to participate in other forms 
of exercise such as walking or resistance training with the upper body.  Compliance with these and 
study dietary requirements will be monitored by subject query at monthly visits during the follow-up 
period.  These visits are also required for dispensing of product from the pharmacy and monitoring 
of consumption compliance. 
a. The study did consider the ethical issue of disallowing resistance training of the thighs during 
the post-training period since the benefits of the exercise training will be partially lost without it.  
We concluded that this criterion was acceptable because it eliminates a strong confounder (i.e. 
strength training) for Aim 3 (Does the supplement promote retention of the benefits of exercise?) 
of the study; older adults are unlikely to participate in high-intensity training of the thighs 
anyway; and other options in exercise more commonly practiced by older adults are acceptable 
as an alternative.         
12. Muscle Biopsy:  Muscle biopsy is a procedure commonly performed in physiology studies.  The 
studies involving this PI have collected several hundred biopsies from the thigh.  Only three 
problems have occurred which involved resumption of bleeding that required a pressure bandage, a 
mild reaction to the lidocaine injection which resulted in halting of the procedure, and fainting which 
led to withdrawal of the subject from the study. 
a. Procedure:  Muscle tissue will be obtained from each subject by needle biopsy of the vastus 
lateralis (outer thigh) by a study physician.  Each subject will undergo the procedure five times.  
The first four will occur before and 72 hrs after a single bout of exercise completed before and 
after approximately 2-weeks of consuming the supplement or placebo.  The final biopsy will 
occur 72 hrs after completion of the exercise training program.  The procedure is performed 
after local anesthetic (lidocaine) is used to numb the site.  A small incision will be made in the 
skin and a Bergstrom needle will be inserted briefly into the muscle to remove several pea-sized 
pieces of muscle (7).  Direct pressure will be applied to stop the bleeding.  The wound is closed 
with a band-aid type bandage and covered with a pressure bandage.  The procedure will be 
performed according to an approved Muscle Biopsy SOP.  The procedure details and outcomes 
will be documented using the Subject Procedure Note.  Pre-exercise biopsies will be taken from 
the dominant leg while post-exercise biopsies will be taken from the non-dominant leg. 
b. Benefit:  The procedure is of no benefit to the subject.  The study will investigate the effects of 
the nutritional supplement on muscle and the response to exercise training and detraining so 
that this information can be used in the future to potentially improve muscle rehabilitation 
strategies.  This information cannot be obtained by alternative procedures.   
c. Risk:  The biopsy procedure may cause discomfort.  Injection of the local anesthetic may sting.  
The discomfort of the biopsy needle insertion varies from painless to uncomfortable or painful.  
The site will be bruised and sore for a few days and will likely cause a small scar.  Temporary 
numbness of the skin at the biopsy site can occur.  Infection of the wound is rare.  Other risks of 
the biopsy include an allergic reaction to the local anesthetic, feeling light-headed or fainting 
during the procedure, or that the wound resumes bleeding later in the day (1:100 cases each). 
d. Minimization of Risk:  The medical history and physical screening process will be used by the 
study physician to exclude subjects that appear to be at increased risk for excessive bleeding or 
have a history of allergic reaction to lidocaine or have had significant problems with fainting.  
*The protocol standard recommendations are that prior to the muscle biopsies subjects should 
refrain from using certain products that can influence bleeding.  Aspirin products and certain 

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supplements should not be used for 10 days prior to the procedure.  Non-aspirin non-steroidal 
anti-inflammatory drugs such as ibuprofen should not be taken for 3 days.  After biopsy, 
subjects will be instructed to refrain from use of these products for 24 hrs though Tylenol may 
be taken if needed.  Subjects will be given a reminder list of the time-frame and products from 
which to refrain.  Subjects will be given verbal and written instructions for caring for the wound 
and extra bandages.  Subjects will be contacted later in the day of the procedure to confirm 
wellness.  If the subject is not reachable, then a message will be left if possible and a second 
attempt will be made later.  The written instructions will contain multiple 24-hr contact numbers 
for the PI and study physicians and describe circumstances under which the subject should 
contact the investigators immediately and/or seek emergency hospital treatment. 
e. *Physician judgement:  Should the requirement for abstinence from contraindicated 
medications and supplements not be adhered to by the subject prior to biopsy, the study 
physician may use clinical judgement to determine the potential for an impact on subject safety 
and the acceptability of undergoing muscle biopsy.  These decisions will be made on a case by 
case basis considering the product consumed, time-frame of consumption, and other individual 
risk factors for bleeding possessed by the subject.    
13. Blood Draw:  Blood draws will be performed by a qualified study staff member or GRECC nurse. 
a. Procedure:  Blood will be drawn nine times over the course of the study from each subject.  
The blood will most likely be drawn from the antecubital vein (≤ 60ml or 2 ounces); however 
other sites may be used at the discretion of the person drawing blood and assent of the subject.  
Fasting is not a requirement.  
b. Benefit:  The purpose of the blood draws are for screening for subject eligibility and safety, and 
for determining the effects of the nutritional supplement on blood-based measures of immune 
function.  If requested, subjects will be provided the results of their clinical blood tests which 
may provide an educational benefit. 
c. Risk:  Blood draw in the proposed amount is a minimal risk procedure.  There is a risk of mild 
discomfort or pain or feelings of lightheadedness.  Fainting is possible.  The blood draw will 
likely cause a small bruise. There is also a small risk of infection. 
d. Minimization of Risk:  The risk of the blood draw will be minimized by the skill of the person 
drawing the blood.  The person drawing blood will utilize standard aseptic technique and follow 
VA policy for hand hygiene and protective coverings.  Sharps will be handled appropriately and 
disposed of properly.  Snacks (e.g. peanut butter crackers and juice) are offered to subjects to 
minimize the feelings of light-headedness after blood draw and biopsy. 
14. Other Human Subject Data Collected 
a. During participation subjects will be asked to maintain their “normal” diet and to not make any 
conscious efforts to lose or gain weight.  Weight will be measured at study entry and upon 
completion. 
b. Use of Certain Medications:  it is unknown how certain medications (specifically those used to 
control inflammation) and other age-associated medical issues will influence the variables of 
interest to the study.  Due to this possibility, medications and medical problems will be recorded 
and used as potential confounding variables in the statistical or exploratory analyses. 
15. Computed Tomography Scan of the Thigh   
a. Procedure:  Muscle Cross-Sectional Area and composition will be determined by the hospital 
imaging service using computed tomography (CT) scan of the mid-thigh at pre-training, post-
training, and at the end of the study.  The service performed CT scans for our previous study 
which showed that size increased (P=0.002, 7.4 ± 2.0%) in older adults after 12-weeks of 

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training [3].  Subjects are carefully positioned in the scanner and boney land marks are used to 
ensure precise mid-thigh location for each scan.  Analysis of scans will be completed using a 
program such as Slice-O-matic (Montreal, Canada) to discern between bone, fat, and muscle. 
CT scan data is transferred from the Imaging service to the study on a CD which is not 
encrypted per se.  However, the risk of an unintended recipient accessing the data is particularly 
low because specialized software is required to view the data.  Furthermore, the data is a cross-
sectional image of the thigh which looks like a slice of ham (i.e. not sensitive).  The information 
will be labeled with the subject study ID, not name or SSN.  Transport of the disk from the LR to 
NLR VA will occur in a locked bag and the disks will be maintained according to CAVHS 
regulations.  
b. Benefit:  The CT scan of the thigh is of no direct benefit to the subject though the measure 
provides important scientific knowledge of the effects of exercise training, detraining, and 
nutritional supplementation in older adults. 
c. Risk:  The procedure will expose subjects to radiation.  The total amount of radiation the 
subjects will be exposed to per scan (6 mSv) is approximately the same as the average 
exposure an individual has from natural sources per year.  Having three scans in one year 
creates a very small increase (from 44.9% to 44.95%) in the risk of cancer for the average 70 
year old male (www.xrayrisk.com).   
d. Minimization of Risk:  There are no exclusion criteria specific to this procedure.  Risk is low in 
the study population as compared to younger populations and individuals who may become 
pregnant. 
16. Data Safety and Monitoring Plan 
a. The following plan has been adapted from "Guidelines for Data and Safety Monitoring for 
Clinical Trials Not Requiring Traditional Data Monitoring Committees” [55]. 
b. The study has eligibility criteria that have been precisely defined, as described above, to ensure 
enrollment of subjects who possess an appropriate risk/benefit ratio. The conservative inclusion 
and exclusion criteria were developed with input from multiple study physicians.  The criteria 
select of individuals with relatively low risk of adverse events from the study procedures and 
relatively high probability of benefit from participation.  These criteria are assessed and 
documented in the Medical History and Physical Form prior to active participation.  Physician 
clearance to participate is documented on this form and CPRS.  
c. Safety monitoring will be performed on a frequent and regular basis from enrollment until at 
least 30 days post-participation for each subject.  Monitoring occurs by CPRS query and subject 
self-report or exam at each research visit as well as during telephone contacts. Thus, during 
participation monitoring per subject will occur at an average rate of more than once per week.   
d. Monitoring will be proportional to the risk of the phase of the study for the individual.  Thus, 
monitoring may occur four times per week per subject if biopsy and exercise are involved but 
less than once per week during post-training follow-up when only nutritional supplementation is 
involved.   
e. Expected adverse events are well-defined as described above and in the informed consent 
document.  All serious adverse events and unexpected adverse events possibly related to 
participation are also monitored for.  The results of monitoring are documented on the submitted 
monitoring checklist as well as in a CPRS note.  This form has both specific expected adverse 
events and open ended questions for unexpected symptoms as well as space for a detailed 
note to chart. 

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f. Appropriate action relative to the severity of the event is taken by the study team immediately 
when an event is identified.  This may range from documentation by the coordinator (e.g. mild 
muscle soreness) to safety examination by the study physician (e.g. elevated blood pressure 
after exercise).  This safety evaluation will include the measures necessary for the study 
physician to judge that the subject can safely continue participation, needs treatment, or should 
be withdrawn from the study. 
g. The study procedures (exercise, supplementation, etc) can and will be halted at any time a 
concern for the subject is identified. Halted procedures will not resume until the concern has 
been alleviated.  If concerns cannot be alleviated then the study physicians may judge to 
withdraw the subject, modify the protocol, suspend enrollment, or halt the study.  This is a 
relatively small single site study which allows these decisions to be made in real-time as they 
occur rather after aggregate data collection after interim analysis.  
h. All adverse events are brought to the attention of the principal investigator and, as needed to 
the study physicians.  Adverse events will be reported to the IRB according to CAVHS policy. 
i. 
The study has also stipulated that a member of the study team will not be blinded to subject 
randomization to supplement or placebo.  This person will not be involved with assessment of 
any outcome measures but will be responsible for monitoring of subject product consumption 
compliance as it relates to the individual, the study, and the pharmacy.  
j. 
The sample size is justified by power calculations presented in this protocol. 
k. The study team members possess no conflicts of interest with regard to the study protocol and 
the procedures and products involved.  
 
17. Data Handling and Recording Plan  
a. The study team has undergone peer-review by the VA Rehabilitation R&D Service to determine 
that the team is composed of appropriately qualified individuals to supervise the overall conduct 
of the trial, to handle the data, to verify the data, to conduct the statistical analyses, and to 
prepare any study reports. 
b. Data collected directly from the subject or their CPRS record is recorded in real-time directly 
onto the submitted data collection forms (e.g. medical history form, exercise training log, 
procedure note) by authorized members of the study team.  Each of these forms has been 
submitted for IRB approval. 
c. These forms are adapted from those used in previous studies and thus are validated for the 
ability to reliably capture the required data. 
d. Data is transferred (recorded) from the data collection forms into an electronic database (Excel 
spreadsheet).  Data entry is performed by a so delegated member of the study team.  The 
process is performed in duplicate (i.e. double data entry) so that the data can be readily 
checked for content validity, completeness, accuracy, and consistency.  Changes to the 
electronic database can be made if needed by the principal investigator or the study team 
member who entered the data. 
e. The database variables will be determined by the principal investigator based on the study 
outcomes collected from or about the subjects per protocol.  Changes to the database variable 
fields will only be made with approval from the principal investigator. 
f. Data collection forms are maintained according to VA research data policy (i.e. indefinitely at 
this time).  Thus, data will not be disposed of or deleted and can be referenced should a 
question arise from the electronic database. 

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g. The paper and electronic records containing HIPAA elements are only accessible to authorized 
members of the study team who are within the VA.  This information is maintained behind 
double locks in the GRECC and on the limited access GRECC server.  This server receives 
frequent backup to ensure safety of content. 
h. Information, paper or electronic, containing HIPAA identifiers will not be shared with the UAMS 
members of the study team or other individuals outside the VA with the exception of potential 
communication between VA and non-VA physicians to ensure subject safety.   
i. 
The electronic database is coded and does not contain HIPAA elements.  The data is 
intentionally transferred without dates to meet this criterion.  The cross-walk file will not leave 
the VA and will not be shared within the VA with any unauthorized individuals.   
j. 
The study has assigned a member(s) of the study team to be not blinded.  This person will 
monitor and record subject compliance and is the study point of contact initial questions 
concerning the supplement/placebo.  The responsibility this study monitor is to safeguard the 
blinding from the other investigators, promote subject compliance, and maintain the integrity of 
the data. 
k. Certain data elements are transformed during transfer from paper to electronic (e.g. birthdate to 
age, subject start and end date to time of participation).  However, it is will always be possible if 
necessary to compare the original data and observations with the processed data.   
l. 
The subject code used in the electronic database is an unambiguous random number that 
allows identification of all the data reported for each subject.  The code is not related to any 
HIPAA element. 
m. It is understood that CAVHS and the VA have ownership of all data.   
n. The study will purchase the investigational products from a VA-authorized vendor and have 
them shipped directly to the VA pharmacy.  The pharmacy will maintain accountability for 
receipt, storage, and dispense of these products as described in the clinical trial product 
information sheet.  The pharmacy and study monitor will also maintain accountability for return 
of unused investigational products. 
 
LABORATORY ASPECTS OF THE STUDY 
Study Outcomes:  The study will examine the effects of the nutritional supplement on measures of 
immune function and muscle health.  The immune response to three types of stimulation will be 
measured:  antibody response to vaccination, immune response in muscle and blood to a bout of 
exercise, and an in vitro response for immune cell proliferation.  Muscle adaptation to training will be 
measured at the whole body (i.e. muscle cross-sectional area, strength, and function) and cellular 
levels.  The blood and muscle tissue collected from the subjects at the described time points will be 
used for laboratory analyses of variables related to immune function and muscle adaptation to exercise 
training.  The basis for the expectations that each measure will be affected by the ingredients of the 
nutritional supplement (β-hydroxy- β-methylbutyrate, arginine, and glutamine) and/or resistance 
exercise is provided.   
The specific proposed method of measurement or measure of immune function (and associated 
hypothesis) in muscle or blood may change for practical and scientific reasons and not be considered a 
deviation so long as the measure:  1) addresses a molecular or cellular trait potentially impacted by 
aging and muscle health or the improvements of muscle health by supplementation or exercise; 2) is 
not genetic testing; 3) and the results will not be communicated back to the subjects or used to 
influence their medical care in any way.    

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BLOOD MEASURES 
A. Response to Vaccination:  β-hydroxy-β-methylbutyrate has been used in animal science to 
improve the response to vaccination [32].  The study will determine if 2-weeks of supplementation 
improves the response of older adults to the TDAP vaccine.  The response to pertussis is of primary 
interest though the responses to the tetanus and diphtheria antigens will also be measured.  Serum 
antibody titers will be compared between pre-vaccination and two time points post-vaccination.  
Enzyme-linked immunosorbent assays are available for measuring antibodies to each antigen (IBL 
International, Toronto, Ontario).   
B. T-Cell Subpopulations:   Glutamine and arginine may be conditionally essential amino acids in the 
contexts of aging and exercise that are needed for optimal T-Cell function [34;56].  Aging decreases 
the ratio of naïve helper (CD4+) to cytotoxic (CD8+) T-Cells [57]; and though the levels of both cell 
populations increase shortly after moderate exercise for older adults, their levels do not reach the 
pre-exercise levels of young adults [58].  The proposed study will determine if 2-weeks of 
supplementation increases the CD4+ to CD8+ ratio or increases the response to acute exercise.  A 
regulatory t-cell subset (CD4+, CD25+, FoxP3+) will also be examined since these cells function as 
regulators of immune function that prevent an uncontrolled immune response or autoimmunity [59].  
Assessment of T-cell subsets will be carried out by flow cytometry using fluorochrome-linked 
antibodies specific to each marker (BD Biosciences, San Jose, CA).  Additionally, if an alteration in 
inflammatory status is indicated by the cytokine analysis, then the Th17 subset (CD4+, CCR6+, 
CD161+, RORα+) will be evaluated.  This pro-inflammatory subset has not been studied following 
supplementation or exercise and the results could provide for the first time information on whether 
alteration in Th17 t-cells accompanies improvement in immune status. 
C. Cytokine and Chemokines:  Immune responses are either predominated by pro-inflammatory Th1 
cytokines (e.g. IFN-γ, TNF-α, IL-2, and IL-17) or anti-inflammatory Th2 cytokines (e.g. IL-10 and IL-
4) and chemokines depending on the nature of the stimulus.  However, arginine and glutamine may 
be able to limit the production of these factors [36].  Since aging is thought to increase basal 
cytokine levels and impair the cytokine response to exercise [60], the study will determine if 2-
weeks of supplementation decreases baseline cytokine levels and increases the response to acute 
exercise.  Cytokines and chemokines will be measured using the multiplex Luminex array system 
(Bio-Rad, Hercules, CA) and confirmed using high sensitivity enzyme linked immunosorbent assays 
(R&D Systems, Minneapolis, MN). 
D. Immune Cell Proliferation:   In vitro models indicate that glutamine and arginine are important to 
immune cell proliferation [34;61].  Similarly, human trials of β-hydroxy- β-methylbutyrate, arginine, 
and glutamine have shown that monocytes, lymphocytes, and T-cells increase after treatment 
[27;46].  Proliferative capacity of lymphocytes also increases shortly after moderate exercise but the 
change is significant only in young and not older adults [58].  This study will determine if 2-weeks of 
supplementation increases the in vitro proliferative capacity of peripheral blood mononuclear cells 
at either baseline or after acute resistance exercise.  After the in vivo effect of the supplement is 
determined, the results will be confirmed using the supplement or its individual ingredients to treat 
cells in vitro.  Proliferation will be measured by flow cytometry based on uptake of a fluorescent dye 
(carboxyfluorescein succinimidyl ester) after treatment with phorbol ester and calcium ionophore as 
was used in Finding 5. 
MUSCLE MEASURES 
E. Macrophages:   Evidence from animal models indicates that arginine and glutamine metabolism is 
involved with macrophage activation that mediates inflammation or anti-inflammation and growth 
[35;37].  Our preliminary results indicate that the majority of macrophages residing in muscle 
perform the latter of these two functions, i.e. anti-inflammation and growth.  However, muscle of 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
Richard Dennis, PhD 
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older adults contains fewer macrophages and the numbers do not increase in response to acute 
exercise.  The study will determine if 2-weeks of supplementation increases the number of 
macrophages present in muscle at baseline or in response to acute resistance exercise.  
Macrophages will be counted in muscle cross section by immunohistochemistry using antibodies to 
detect the CD163 marker (Cell Sciences, Canton, MA).  Additional markers (CD68 and CD206) will 
be used to confirm the specificity of the CD163 marker to macrophages. 
F. Cytokines and Growth Factors:   Our preliminary results show that older muscle contains 
elevated basal levels of cytokines and diminished levels of growth factors.  The effect of arginine 
and glutamine on cytokine expression is expected to be the same in tissue as it is systemically.  
The study will determine if 2-weeks of supplementation decreases baseline cytokine levels and 
increases the response to exercise.  Since chronic inflammation is thought to dampen growth factor 
production [62], the study will determine if supplementation also increases growth factor levels at 
baseline and in response to exercise.  IL1β, IL10, and IGF1 are of primary interest though the other 
cytokines, and growth and remodeling factors including MuRF and MAFbx which will also be 
measured.  Gene expression will be measured at the mRNA level using total RNA extracted from 
whole muscle by quantitative real-time PCR.  Assays will utilize SYBR green chemistry and 
standard curves as was used in Findings 1-4 (Figures 2-5). 
G. Nuclear Factor-kB (NFkB) and Phosphatidylinositol 3-Kinase Signaling (PI3K):   Direct 
assessment of protein and protein activity in human muscle has proven difficult for cytokines and 
growth factors including IGF1, one of the most commonly studied regulators of muscle mass.  For 
this reason, the study will evaluate muscle for cytokine (NFkB)  and growth factor (PI3K) activity by 
evaluating signal transduction pathways involved in regulation of muscle hypertrophy and atrophy 
[63].  The study will determine if 2-weeks of supplementation decreases NFkB signaling and 
increases PI3K signaling at baseline and in response to exercise.  NFkB activity will be assessed by 
electrophoresis mobility shift assays to detect muscle nuclear protein binding to the NFkB DNA site 
as used in Figure 9A or as follows for PI3K.  PI3K activity will be assessed by Western Blot based 
phosphorylation assays (Millipore, Billerica, MA). 
H. Additional Signaling Pathway Members:  The regulation of inflammation and growth in 
macrophages and muscle cells is regulated by a complex network of signaling molecules.  Thus, in 
addition to NFkB and PI3K, other factors will also be measured for effects of supplementation and 
exercise [24;25;64].  Of main interest are FOXO1, STAT3, AKT, S6K, and ERK1 and 2.  The activity 
of these factors is regulated, in part, by their phosphorylation state.  Matched antibody pairs are 
available to measure the ratio of these phosphorylated to un-phosphorylated proteins by Western 
Blot (Cell Signaling Technology, Boston, MA).  Pathway scanning kits are also available which 
include other related molecules in the cascade.  Muscle remains from the nutritional supplement 
pilot study presented in the introduction.  This muscle will enable an initial broad assessment of the 
signaling pathways affected by nutritional supplementation.  The results will guide any changes in 
the specific factors chosen for assay in muscle collected for the proposed study.  
I. Satellite Cells and Myonuclei:   Muscle adapts to resistance training through increases in satellite 
cell numbers and addition of nuclei to the myofiber [21].  However, these adaptations are 
diminished in older adults [22].  The study will determine if 15-weeks of supplementation increases 
the number of satellite cells and myonuclei per myofiber in muscle.  Satellite cells (Pax7+, CD56+, 
DAPI+) and myonuclei (Dystrophin plus DAPI staining) will be counted using established staining 
methods [21]. 
J. Fiber Size:  Muscle also adapts to resistance training by muscle fiber hypertrophy.  However, this 
growth is hindered in individuals, particularly older adults, who have a diminished satellite cell and 
myonuclei response to training [22].  The study will determine if 15-weeks of supplementation 
increases the effects of resistance training on myofiber size.  Changes in fiber size will be 

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measured microscopically as the average percent increase in fiber cross-sectional area from 
baseline to post-training. 
 

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STATISTICAL PLAN:  The sample 
size (N=50) provides greater than 
80% power for Aims 1-3 to detect 
mean differences between groups of 
0.8 to 0.85 standard deviation (SD) 
units at a 5% level of significance.  
Power calculations were based on 
using a one-sided t-test to compare 
independent groups using data from 
our work and others [2;23;65] though 
non-parametric tests or 
transformations will be used as 
necessary for data containing 
outliers.  There is no formal plan to 
adjust for multiple comparisons.  
However, multiple correlation 
analysis will be used to determine 
the correlation between endpoints and any possible inflation in the overall Type I error rate.  This 
analysis will allow testing of the overall hypothesis that measures of immune function (Aim 1) will be 
positively correlated with adaptations to exercise (Aim 2) and retention of muscle strength, mass, and 
function after exercise training is halted (Aim 3).  The hypotheses are stated for each aim and the time 
points for comparison are listed in Table 6. 
Aim 1 Power:  Aim 1 will determine if the supplement consumption for 2-weeks improves baseline 
measures of immune function or the immune responses to vaccination or acute exercise.  The 
representative measures chosen to calculate power are change in muscle macrophage content after 
exercise (Finding 2) and change in CRP levels post-supplementation (Pilot Study Finding 1).  Przybyla 
et al detected a 29% increase in CD163+ macrophages in young subjects after exercise but no change 
in older subjects [2].  Power for this aim is calculated to detect at least a 29% greater increase in 
macrophages in the supplement than the placebo group.  Przybyla et al used a nonparametric test to 
lessen the effects of outliers.  To be more conservative here, the calculations here dropped the most 
extreme high and low data values before estimating effect sizes.  With that approach, the 29% increase 
corresponds to an effect size of 0.8 SD units (mean/SD = 0.128/0.159).  Similar results were found 
using CRP data from Pilot Finding 1.  The effect size for change in CRP is 0.85 SD units (mean/SD = 
0.665/0.780 for log-transformed data).  Having at least 20 subjects per group will provide 80% or 
greater power to detect a mean shift of 0.8-0.85 SD units at a significance level of 5% for the following 
Aim 1 hypotheses: 
a. The response to vaccination will be greater in the supplement than the placebo group.  The change 
between times K and A (Table 6) will be compared between groups. 
b. The resting ratio of CD4+ to CD8+ t-cells will increase in the supplement but not in the placebo 
group.  The change between times G and C will be compared between groups. 
c. The change in t-cell subsets after acute exercise will be greater in the supplement than the placebo 
group.  The change between times I and G will be compared to the change between times E and C 
for both groups.  
d. Resting cytokine levels will decrease in the supplement but not in the placebo group.  Change will be 
compared between groups for blood (times G and C) and muscle (times H and D).  Similar results 
are predicted for the MuRF and MAFbx atrophy-related transcripts. 
e. The increase in cytokine levels after acute exercise will be greater in the placebo group than the 
supplement group.  For both groups, change between times I and G will be compared to the change 
Table 6.  Time points are lettered for clear reference by the stated hypotheses.  
BASELINE MEASURES (WEEKS 1-5) 
A. 
Blood draw at study enrollment for measuring pre-vaccination antibody titers 
B. 
Assessment of muscle strength, mass, and function at baseline 
C. 
Blood drawn prior to acute exercise at baseline 
D. 
Muscle biopsy prior to acute exercise at baseline 
E. 
Blood drawn 1 hr after acute exercise at baseline 
F. 
Muscle biopsy 72 hrs after acute exercise at baseline 
POST-SUPPLEMENT RUN-IN MEASURES (WEEK 8) 
G. 
Blood drawn prior to acute exercise after 2-weeks of supplementation  
H. 
Muscle biopsy prior to acute exercise after 2-weeks of supplementation 
I. 
Blood drawn 1 hr after acute exercise after 2-weeks of supplementation 
J. 
Muscle biopsy 72 hrs after acute exercise after 2-weeks of supplementation 
MEASURES DURING TRAINING PERIOD (WEEKS 9-20) 
K. 
Blood draws at weeks 1 and 2 post-vaccination for measuring antibody response 
POST-TRAINING MEASURES (WEEK 21) 
L. 
Assessment of muscle strength, mass, and function after completion of training  
M. 
Muscle biopsy 72 hrs after final exercise bout of training 
MEASURES DURING POST-TRAINING FOLLOW-UP (WEEKS 22-4) 
N. 
Assessments of strength, mass, and function after 16 and 26 weeks of detraining 

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between E and C for blood, and for muscle the change between times J and H will be compared to 
the change between times F and D. 
f. The proliferative capacity of peripheral blood mononuclear cells will be greater in the supplement 
than the placebo group.  The change between times G and C will be compared between groups.  
The change between times I and E will also be compared between groups. 
g. The resting number of muscle CD163+ macrophages will increase in the supplement but not in the 
placebo group.  The change between times H and D will be compared between groups. 
h. The number of muscle CD163+ macrophages will increase after acute exercise in the supplement 
but not in the placebo group.  The change between times J and H will be compared to change 
between times F and D for both groups. 
i. Resting NFkB activity will decrease and PI3K activity will increase in the supplement group but not in 
the placebo group.  The change between time points H and D will be compared between groups. 
j. In response to acute exercise, NFkB activity will be lower and PI3K activity will be greater in the 
supplement group than in the placebo group.  The change between times J and H will be compared 
to change between times F and D for both groups.  Responses similar to NFkB are predicted for 
FOXO1, and responses similar to PI3K are expected for STAT3, AKT, P38 MAPK, and ERK 1 and 2.   
Aim 2 Power:  Aim 2 will determine whether supplement consumption increases muscle adaptation to 
12-weeks of resistance training.  The representative response measure for this objective is post-training 
percent increase in muscle strength.  In a similar study, Trappe et al sought to improve strength gain by 
resistance training plus drug treatment.  The drug treatment group gained significantly more strength by 
25.1% than the group receiving placebo (26.4% vs. 21.1%, P<0.05) [23].  The previous findings 
presented here (Finding 4) indicate that older subjects completing a 12-week training program but not 
taking supplements will experience an average strength gain of 25 ± 8%.  If treatment with the 
proposed supplement improves strength similarly to the Trappe drug treatment, subjects in the 
supplement group will improve strength by an average of 31% (i.e., 25.1% greater than those of the 
placebo group).  This translates to an approximate effect size of 0.8 SD units (0.25*0.251/0.08) if 
variances are pooled for the groups compared.  With 20 subjects per group, there will be 80% power to 
detect a mean shift of 0.8 SD units using a significance level of 5% for the Aim 2 hypotheses: 
a. The amount of strength gained from 12-weeks of training will be greater in the supplement than the 
placebo group.  The change between times L and B will be compared between groups. 
b. The amount of muscle size gained from 12-weeks of training will be greater in the supplement than 
the placebo group.  The change between times L and B will be compared between groups. 
c. The increase in muscle function after 12-weeks of training will be greater in the supplement than the 
placebo group.  The change between times L and B will be compared between groups. 
d. The increase in muscle satellite cells after 12-weeks of training will be greater in the supplement 
than the placebo group.  The change between times M and D will be compared between groups. 
e. The increase in muscle myonuclei after 12-weeks of training will be greater in the supplement than 
the placebo group.  The change between times M and D will be compared between groups. 
f. The increase in muscle fiber size after 12-weeks of training will be greater in the supplement group 
than the placebo group.  The change between times M and D will be compared between groups. 
Aim 3 Power:  Aim 3 will determine if supplement consumption increases the retention of exercise-
derived benefits post-training during 26 weeks of long-term follow-up.  The representative response 
measure for this objective is the strength gain retained during long-term follow-up after exercise training 
is completed.  Lemmer et al observed an average gain in muscle strength of 27% (P<0.01) in older 
men after strength training which is similar the 25% gain presented here in Finding 4 [65].  However, at 

Immune Function and Muscle Adaptations to Resistance Exercise in Older Adults 
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V11, 06-26-2017 
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31-weeks of post-training follow-up, strength had decreased to be about 9% above pre-training levels.  
Assuming the same level of variation among our subjects in retention of strength gain as the observed 
8% SD for strength gain in Finding 4, then a 0.8 SD effect size would be about 6.4% (0.8*0.08), 
translating to our supplement group retaining strength of 12.4% above pre-training levels.  With 20 
subjects per group, there will be 80% power to detect a mean shift of 0.8 SD units using a significance 
level of 5%.  If the SD for retention of strength is smaller than 8%, then we will have higher power to 
detect the same difference or equal power to detect smaller differences for the following Aim 3 
hypotheses: 
a. The change in strength during the detraining period will be smaller in the supplement than the 
placebo group.  The change between times N and L will be compared between groups. 
b. The change in muscle size during the detraining period will be smaller in the supplement than the 
placebo group.  The change between times N and L will be compared between groups.   
c. The change in muscle function will be smaller in the supplement than the placebo group. The 
change between times N and L will be compared between groups. 
 

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