The Effects of Varying Essential Amino Acid Intakes on Resting and Post-exercise Skeletal Muscle and Whole-body Protein Kinetics During Negative Energy Balance
Phase: NA
Conditions: Muscle Protein Synthesis, Whole-body Protein Balance
Sponsor: United States Army Research Institute of Environmental Medicine

Cover Page 
 
PROTOCOL TITLE:  The effects of varying essential amino acid intakes on resting and post-exercise skeletal 
muscle and whole-body protein kinetics during negative energy balance 
 
NCT#: 03372928 
 
Document Dates: 15JAN19 
 
 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
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PROTOCOL TITLE:  The effects of varying essential amino acid intakes on resting and post-exercise skeletal 
muscle and whole-body protein kinetics during negative energy balance 
 
SECTION A:  RESEARCH TEAM AND LOCATIONS 
 
A1.  RESEARCH TEAM 
 
Study Role 
Institution/Company and Contact Information 
Sponsor 
Organization/Institution/Company:   
Military Nutrition Division (MND), US Army Research Institute 
of Environmental Medicine (USARIEM) 
Address: 10 General Greene Ave, Bldg. 42 Natick, MA 01760 
Point of Contact:  N/A 
 
Principal Investigator 
Name and Degree: Stefan M Pasiakos, PhD 
Title:  Nutritional Physiologist 
Institution:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-6474 
Email:  stefan.m.pasiakos.civ@mail.mil 
 
 
Associate Investigator(s) 
 
Name and Degree: Arny A Ferrando, PhD 
Title:  Professor  
Institution/Company: University of Arkansas for Medical 
Sciences (UAMS) 
Address:  4301 West Markham St, Slot 806 
Phone Number: 501-526-5711 
Email:  AFerrando@uams.edu 
Name and Degree:  Lee M Margolis, PhD, RD 
Title: ORISE Post-doctoral fellow  
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 30 Natick, MA 01760 
Phone Number:  508-233-4291 
Email:  lee.m.margolis.ctr@mail.mil 
Name and Degree:  Jess A Gwin, PhD 
Title: ORISE Post-doctoral fellow  
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 30 Natick, MA 01760 
Phone Number:  508-233-4794 
Email:  jessica.a.gwin.ctr@mail.mil 
Name and Degree:  David D Church, PhD 
Title: UAMS Post-doctoral fellow  
Institution/Company:  University of Arkansas for Medical 
Sciences (UAMS) 
Address:  4301 West Markham St, Slot 806 
Phone Number:  501-526-5735 
Email: DChurch@uams.edu 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
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Consultant(s) 
 
 
 
 
 
 
 
Other Key Research  
Personnel (as applicable) 
 
Name and Degree:  John W Carbone, PhD, RD 
Title:  Associate Professor 
Institution/Company: Eastern Michigan University 
Address: 312 Everett L Marshall Bldg., Ypsilanti, MI 48197 
Phone Number: 734-487-4096 
Email:  jcarbon2@emich.edu 
Name, Rank, and Degree:  Adrienne Hatch, MS, RD 
Title:  Project Coordinator  
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 30 Natick, MA 01760 
Phone Number:  508-233-5648 
Email:  adrienne.m.hatch.civ@mail.mil 
 
 
Other Individuals 
Supporting  
the Research (as 
applicable) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name, Rank, and Degree:  Nancy Murphy, MS 
Title:  Laboratory Manager 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4598 
Email:  nancy.e.murphy5.civ@mail.mil 
Name, Rank, and Degree:  Christopher Carrigan, BS 
Title: Research Physiologist  
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5101 
Email: christopher.t.carrigan.civ@mail.mil 
 
Name, Rank, and Degree:  Marques Wilson, MS  
Title:  Research Physiologist 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5681 
Email:  marques.a.wilson.civ@mail.mil 
 
Name, Rank, and Degree:  Claire Whitney, MS, RD 
Title:  Research Dietitian,  
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4875 
Email: claire.c.whitney.civ@mail.mil 
 
Name, Rank, and Degree:  Heather S. Fagnant, MS, MPH, 
RD, LDN 
Title:  Research Dietitian, ORISE Fellow  
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-233-5958 
Email: heather.s.fagnant.ctr@mail.mil 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
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Page 4 of 31 
 
 
 
 
 
 
 
 
 
Name, Rank, and Degree:  Anthony Karis, BS 
Title:  Research Physical Scientist 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 30, Natick, MA 01760 
Phone Number:  508-233-4754 
Email:  anthony.j.karis.civ@mail.mil 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name, Rank, and Degree:  SGT Alfonzo Patino 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4715 
Email:  alfonzo.m.patino.mil@mail.mil 
Name, Rank, and Degree:  SGT Brandon Cordell 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5958 
Email:  brandon.j.cordell.mil@mail.mil 
Name, Rank, and Degree:  SPC Katakyie Sarpong, BS 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4835 
Email:  katakyie.p.sarpong.mil@mail.mil 
Name, Rank, and Degree:  SPC Jason Alaniz 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  361-271-8105 
Email:  jason.alaniz3.mil@mail.mil 
Name, Rank, and Degree:  SPC Marcus Sanchez 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number:  (508) 397-5911 
Email:  marcus.a.sanchez20.mil@mail.mil 
Name, Rank, and Degree:  SGT Cassandra Rousayne, MS 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5140 
Email:  cassandra.l.rousayne.mil@mail.mil 
 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
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Research Monitor 
 
 
 
 
 
 
 
Ombudsmen                           
Name, Rank, and Degree:  SSG Stephen Mason 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  540-718-4446 
Email:  stephen.a.mason14.mil@mail.mil 
Name, Rank, and Degree:  Patrick Radcliffe 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number: n/a 
Email:  patrick.n.radcliffe.ctr@mail.mil 
Name, Rank, and Degree:  Emily Howard 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  n/a 
Email:  emily.e.howard14.ctr@mail.mil 
Name, Rank, and Degree:  Jillian Allen, MS, RD 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  617-240-3838 
Email:  n/a 
Name, Rank, and Degree: MAJ Robin E. Cushing, DrPH, PA-C 
Title:  Medical Monitor 
Institution/Company:  Office of Medical Support and 
Oversight, USARIEM 
Address:  10 General Greene Ave Bldg. 86, Natick, MA 01760 
Phone Number: 508-233-5128 
Email: robin.e.cushing.mil@mail.mil 
Name, Rank, and Degree:  Katelyn Guerriere, MS 
Title:  Research Physiologist 
Institution/Company:  MPD, USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 
01760 
Phone Number: 508-233-5619 
 Email:  Katelyn.i.guerriere.civ@mail.mil 
Name, Rank, and Degree:  Katherine Mitchell, MS 
Title:  Research Physiologist 
Institution/Company:  TMMD, USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 
01760 
Phone Number: 508-233-4177 
 Email:  katherine.m.mitchell15.civ@mail.mil 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
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 Name, Rank, and Degree: Karleigh Bradbury, MS 
Title:  Research Physiologist 
Institution/Company:  TMMD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 
01760 
Phone Number: 508-233-4977 
 Email:   karleigh.e.bradbury.civ@mail.mil 
Name, Rank, and Degree: Caitlin Haven, MS 
Title: Research Statistician 
Institution/Company: MPD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick MA 01760 
Phone Number: 508-233-4853 
Email: Caitlin.c.haven.civ@mail.mil 
Name, Rank, and Degree: Maria Canino, MS 
Title: ORISE Research Fellow 
Institution/Company: MPD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick MA 01760 
Phone Number: 708-253-7983 
Email: maria.c.canino.ctr@mail.mil 
Name, Rank, and Degree: Matthew Bartlett, BS 
Title: ORISE Research Fellow 
Institution/Company: MPD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick MA 01760 
Phone Number: 781-606-1819 
Email: paul.m.bartlett5.ctr@mail.mil 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
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A2.  ROLES AND RESPONSIBILITIES 
      
A2.1 Key Research Personnel 
 
Name(s):  Stefan M Pasiakos 
Research Role:  Principal Investigator 
Study Responsibilities:  The Principal Investigator is responsible for the safe and scientifically sound conduct of 
the study.  He will oversee all aspects of the study, ensure safety and ethical treatment of volunteers, maintain 
required documentation for the study and obtain required approvals, and will have primary responsibility for 
data analysis, interpretation, and publication.  Dr. Pasiakos will be involved in volunteer briefing, obtaining 
informed consent, data collection, performing muscle biopsies, catheterization, phlebotomy, exercise testing, 
and interventions.  
 
Name(s):  Arny A Ferrando 
Research Role:  Associate Investigator 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, hypotheses, 
experimental approach, and design. Assist PI with data analysis, particularly stable isotope assessments of 
skeletal muscle and whole-body protein kinetics.  Dr. Ferrando will receive and analyze coded, de-identified 
blood and muscle samples for isotopic analysis.  Dr. Ferrando will also be actively involved with the 
interpretation of the data and preparation of peer-reviewed manuscripts. He will observe test volunteers and 
the study process to ensure correct study implementation, but will not have access to personal identifiable 
information.  A coded specimen transfer agreement is included with this submission.  
 
Name(s):  Lee M Margolis 
Research Role:  Associate Investigator 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, hypotheses, 
experimental approach, and design. Assist PI with data collection, management, and analysis and manuscript 
preparation. Prepare and administer test diets. Data collection will involve percutaneous skeletal muscle 
biopsies, phlebotomy, catheterization, monitoring submaximal exercise testing, and sample processing. In the 
event of Dr. Pasiakos’ absence, Dr. Margolis can oversee, manage, and execute all study procedures, 
particularly the stable isotope infusion and muscle biopsy studies.  
 
Name(s):  Jess A Gwin 
Research Role:  Associate Investigator 
Study Responsibilities:  Assist PI with data collection, management, and analysis and manuscript preparation. 
Prepare and administer test diets. Data collection will involve percutaneous skeletal muscle biopsies, 
phlebotomy, catheterization, monitoring submaximal exercise testing, and sample processing.  Dr. Gwin can 
execute the stable isotope infusion and muscle biopsy studies.  She will also be actively involved with the 
interpretation of the data and preparation of peer-reviewed manuscripts and technical reports for publication. 
 
Name(s):  David C Church 
Research Role:  Associate Investigator 
Study Responsibilities:  Assist with data collection, biological sample processing, and biological sample 
analyses.  David Church will not be involved in any credential requiring study processes.   
 
Name(s):  John W Carbone 
Research Role:  Consultant 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, hypotheses, 
experimental approach, and design. Assist PI with data interpretation and manuscript preparation. Dr. Carbone 
will not receive coded data, nor be engaged in this collaborative research project.  He will not interact or 
intervene with test volunteers, or have access to personal identifiable information. 
 
 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
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Name(s):  Adrienne Hatch 
 
Research Role:  Project Coordinator, Study Dietitian 
Study Responsibilities:  Supervise, manage, and coordinate study logistics and biological data collection. She 
will be involved with protocol development, menu development, volunteer briefing, preparation and 
administering study diets to volunteers, diet instruction, dietary assessment, and study implementation. She 
will actively participate in data collection to include phlebotomy, monitoring submaximal exercise testing, and 
interventions, and DEXA. 
 
A2.2. Others Involved in the Research, as applicable  
 
 
Name(s):  Claire Whitney, Heather Fagnant, Jillian Allen 
 
Research Role:  Research Dietitians 
Study Responsibilities:  Menu development, preparation and administering study diets to volunteers, diet 
instruction, dietary assessment, and study implementation. Claire Whitney and Heather Fagnant will assist with 
DEXA. Claire Whitney will additionally assist with phlebotomy, monitoring submaximal exercise testing, and 
interventions. 
 
Name(s):  Nancy E Murphy 
Research Role:  Biological Sample Coordinator 
Study Responsibilities:  Supervision, management, and coordination of logistics, and biological data collection. 
She will be involved with protocol development and study implementation.  Data collection responsibilities will 
involve sample processing, management, and oversight, and DEXA.  She will be responsible for study 
randomization management.  
 
Name(s):  Christopher T Carrigan 
Research Role:  Research Physiologist 
Study Responsibilities:  Assist with data collection, phlebotomy, catheterization, DEXA, and biological sample 
processing.  
 
Name(s):  Marques Wilson 
Research Role:  Research Physiologist 
Study Responsibilities:  Assist with data collection and biological sample processing.  Marques Wilson will also 
be involved in phlebotomy, catheterization, DEXA, and administration of maximal muscle exercise testing and 
intervention 
 
Name(s):  SPC Alaniz, SGT Cordell, SGT Patino, SPC Sarpong, SPC Sanchez, SSG Mason, SGT Rousayne, 
Patrick Radcliffe, Emily Howard, and Anthony Karis 
Research Role:  Research Assistants 
Study Responsibilities:  Assist with data collection, phlebotomy, and biological sample processing. SGT Cordell 
and SGT Patino may also assist with catheterization.  Patrick Radcliffe and Emily Howard will not perform 
phlebotomy. 
 
Name(s):  MAJ Robin Cushing 
Research Role:  Research Monitor  
Study Responsibilities:  The research monitor shall review all unanticipated problems involving risk to subjects 
or others, serious adverse events and all subject deaths associated with the protocol and provide an unbiased 
written report of the event. 
 
Name(s):  Katelyn Guerriere, Katherine Mitchell, Karleigh Bradbury, Maria Canino, Caitlin Haven, Matthew 
Bartlett   
Research Role:  Ombudsman 
Study Responsibilities:  Observe group briefings for military volunteers not in the Human Research Volunteer 
program.  

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
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A3.  RESEARCH LOCATIONS    
 
USARIEM, Natick MA: USARIEM is a DoD research facility within the US Army Medical Research and Materiel 
Command.  It is the Institute responsible for conducting basic and applied research to determine the effects of 
exposure to environmental extremes, occupational tasks, physical training, deployment, operational stress and 
nutritional factors on the health and performance of military personnel.  The facility contains environmental 
chambers for controlling temperature and humidity, an environmentally controlled hypobaric chamber, a water 
immersion laboratory, as well as several dry and wet laboratories for animal and human experimentation.  The 
dry laboratories are capable of a broad range of experiments, including biomechanical analysis, body 
composition, energy expenditure, and muscle strength and endurance.  The wet laboratories include general 
clinical chemistry analyzers, as well as equipment for ELISA, RIA, histology, and molecular biology assays.  
Each investigator at the facility has a personal computer with software for data management, analysis, 
presentation and report generation.  Staff computers are interfaced with a network server for easy, secure data 
handling and transfer.  All testing (pre-study screening, baseline, and experimental testing) will take place at 
USARIEM. 
 
UAMS, Little Rock, AR: The Center for Translation Research in Aging & Longevity (CTRAL), Donald W 
Reynolds Institute of Aging (RIOA), University of Arkansas for Medical Science (UAMS) has the basic 
laboratory facilities and equipment to analyze stable isotope kinetics, including 2 Agilent 5973 GC/MS, and 3 
Agilent 5975 GC/MS, a Finnegan TSQ 7000 LC/MS/MS, and a Waters QTOF LC/MS.  The CTRAL is led by 
the world’s foremost expert in stable isotope assessments of human metabolism, Dr. Robert R Wolfe.  
 
 
SECTION B:  RESEARCH METHODOLOGY 
 
B1.  ABSTRACT   
 
Short-term negative energy balance (i.e., energy deficit) downregulates muscle protein synthesis and 
upregulates whole-body proteolysis and amino acid (AA) oxidation, thereby increasing nitrogen excretion and 
exacerbating whole-body and skeletal muscle protein loss.  Consumption of quality proteins high in essential 
amino acid (EAA, i.e., the active anabolic component of protein) content may attenuate protein loss during 
energy deficit by restoring whole-body and skeletal muscle anabolic potential to that observed in a eucaloric 
state.  During energy balance, muscle protein synthesis appears to be maximally stimulated after consuming 
15 g of EAA at rest and after conventional resistance-type exercise.  In response to a short-term energy deficit 
that downregulated basal muscle protein synthesis by as much as 27%, consuming 15 g (~7.5 g EAA) and 30 
g (~15 g EAA) of whey protein after a bout of resistance exercise restored muscle protein synthesis rates to 
resting, fasted rates observed in the eucaloric state in a dose dependent manner.  The effect of EAA intakes 
above 15 g on resting and post-exercise muscle protein synthesis and the whole-body protein anabolic 
response during acute energy deficit has not been determined.  To address this, we will assess resting and 
post-resistance exercise whole-body and skeletal muscle protein synthesis responses to across a spectrum of 
EAA intakes following a well-controlled, short-term (5-d) energy deficit (30% energy deficit).  Using a 
randomized, double-blind, cross-over design, 20 resistance trained (≥ 2 d/wk for the past 6 mo) adults will 
undergo two, non-consecutive 5-d energy deficit periods, separated by a 14-d washout period.  Resting and 
post-resistance exercise (single leg exercise model) whole-body protein turnover and skeletal muscle protein 
synthesis responses to two different doses of EAA (standard, 0.10 g/kg vs high, 0.30 g/kg) will be determined 
the morning after completing the 5-d energy deficit. This design will test the hypothesis that higher absolute 
doses of EAA are required to maintain resting and post-exercise anabolic responses during energy deficit.  
Risks include those associated with venous catheterization, muscle biopsies, DEXA, exercise, and the 
discomfort of moderate underfeeding.   
 
B2.  BACKGROUND AND SIGNIFICANCE  
Periods of negative protein balance (i.e., breakdown > synthesis) are largely unavoidable during strenuous 
military operations, and is exacerbated by sub-optimal dietary protein and energy intake [1, 2]. Prolonged 

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Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
Page 13 of 31 
 
joint effort between USARIEM and the US Army Combat Feeding Directorate, i.e., the Army Science and 
Technology Objective: Nutritionally Optimized Food Products for an Expeditionary Force (R.MRMC.2017.01). 
B4.  OBJECTIVES/SPECIFIC AIMS/RESEARCH QUESTIONS   
 
Objectives 
 
1. Characterize effects of essential amino acid (EAA) quantity on resting and post-resistance exercise 
muscle and whole-body protein kinetic responses during short-term energy deficit. 
 
2. Determine the effects of essential amino acid (EAA) quantity on resting and post-resistance 
exercise skeletal muscle anabolic signaling during short-term energy deficit. 
 
Hypotheses 
 
1. Resting and post-resistance exercise muscle protein synthesis will be stimulated to a greater 
degree with higher (0.30 g/kg) versus lower (0.10 g/kg) EAA intake during acute, moderate energy 
deficit. 
 
2. Resting and post-resistance exercise whole-body protein balance will be greater with higher (0.30 
g/kg) versus lower (0.10 g/kg) EAA intake during acute, moderate energy deficit. 
 
3. Resting and post-resistance exercise whole-body protein balance will be more positive with higher 
(0.30 g/kg) versus lower (0.10 g/kg) EAA intake during acute, moderate energy deficit due to a 
greater increase in whole-body protein synthesis.   
 
4. Resting and post-resistance exercise skeletal muscle anabolic signaling will be stimulated to a 
greater degree with higher (0.30 g/kg) versus lower (0.10 g/kg) EAA intake during acute, moderate 
energy deficit. 
 
B5.  RESEARCH PLAN   
 
B5.1 Research Design   
 
This study will be a randomized, double-blind, cross-over controlled trial. Before the study begins, a research 
team member (Nancy Murphy) will generate the randomization scheme using a random number generator 
(http://randomization.com).  Ms. Murphy will be responsible for preparing and coding the individualized EAA 
drinks for each trial and she will hold the randomization code until the study has been completed (i.e., data 
collected and analyzed).  The study investigators will be blinded to this process.  The code will be broken by 
Ms. Murphy after the primary outcomes (i.e., muscle protein synthesis and whole-body protein balance) have 
been statistically analyzed by the study investigators (i.e., Pasiakos, Ferrando, Margolis, Gwin, Church, and 
Carbone).  
 
B5.2 Research Subjects/Population(s) 
 
B5.2.1 Subject Population(s)   
 
Twenty healthy, recreationally-active adult men and women representative of active duty military 
service members will be tested in this study. This study will recruit up to 70 individuals in order to 
complete testing on 20. These individuals may be recruited from the HRV population, civilians, and 
active duty military personnel (on and off the installation). 
 
B5.2.2 Number of Subjects, Records, and/or Specimens   
 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
Page 14 of 31 
 
In previous studies of similar design, pre-study screening failure rate is approximately 25%.  To account 
for screening failures and the possibility that actual study attrition rates may be greater, we estimate 
that 30 individuals will require screening in order to complete testing on the 20 volunteers necessary to 
reach statistical power in our primary outcomes.  All screening will stop once complete data has been 
collected on 20 volunteers.  Records and specimen collection are described in the Research 
Procedures and Data Collection sections.  During briefings and consenting, potential participants will be 
informed that even though they may be eligible and want to participate, if we are able to obtain enough 
data from preceding subjects, they may not ultimately be tested.      
 
B5.2.3 Inclusion Criteria 
 
• 
Men and women aged 18 – 35 years 
• 
Body mass index < 30.0 kg/m2 
• 
Healthy without evidence of chronic illness or musculoskeletal injury as determined by the 
USARIEM Office of Medical Support and Oversight (OMSO) 
• 
Resistance exercise trained defined by self-report as performing ≥ 2 sessions/wk for previous 6 mo 
• 
Refrain from taking any nonsteroidal anti-inflammatory drugs (e.g., aspirin, Advil®, Aleve®, 
Naprosyn®), or any other aspirin-containing product for 10 days before starting and at least 5 days 
after completing the study 
• 
Willing to refrain from alcohol, smoking any nicotine product (includes e-cigarettes); vaping, 
chewing tobacco, caffeine, and dietary supplement use throughout the entire study period  
• 
Supervisor approval for federal civilian employees and non-HRV active duty military personnel 
working within the US Army Natick Soldier Systems Center 
 
B5.2.4 Exclusion Criteria 
 
• 
Musculoskeletal injuries that compromise exercise capability as determined by the USARIEM Office 
of Medical Support and Oversight (OMSO) 
• 
Metabolic or cardiovascular abnormalities, gastrointestinal disorders (e.g., kidney disease, diabetes, 
cardiovascular disease, etc.)  
• 
Abnormal PT/PTT test or problems with blood clotting  
• 
History of complications with lidocaine  
• 
Present condition of alcoholism, anabolic steroids, or other substance abuse issues 
• 
Blood donation within 8-wk of beginning the study 
• 
Pregnancy (self-report or results of urine pregnancy test before body composition testing) 
• 
Unwillingness or inability to consume study diets or foods provided 
 
B5.3 Research Procedures   
 
Experimental Design 
Twenty resistance-trained adults will be tested in this approximately 39-d randomized, double-blind cross-over 
study.  Volunteers will participate in two, non-consecutive 5-d controlled energy deficit periods, separated by a 
14-d washout period (i.e., sufficient time to restore nitrogen and metabolic homeostasis after moderate weight 
loss) [35-38].  Basal (fasting/resting), resting and post-resistance exercise (single leg exercise model) whole-
body protein turnover and skeletal muscle protein synthesis responses to standard (0.10 g/kg) and high (0.30 
g/kg) EAA doses will be determined the morning after completing each 5-d energy deficit. To limit potential 
confounding effects of pre-study diet, and to be consistent with our previous research [16, 39, 40], volunteers 
will be provided a 3-d run-in, eucaloric diet.  After the 3-d eucaloric run-in diet, energy intake will be reduced by 
30%.  Physical activity will be completely restricted during the 3-d run-in diet and 5-d energy deficit to isolate 
the effects of the energy deficit on protein turnover and limit any residual effects of previous exercise on 
outcome measures [41].  
 

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The 30% energy deficit from diet alone is consistent with our previous energy deficit studies [42] and work 
demonstrating that a 10-d, 20% diet-induced energy deficit (i.e., ~500 kcal/d) downregulates resting skeletal 
muscle protein synthesis by as much as 19% [43].  Others have shown that 5-d of moderate energy deficit 
(reducing energy availability from 50 kcal/kg fat-free mass to 30 kcal/kg fat-free mass) is long enough to 
downregulate resting skeletal muscle protein synthesis by 27% [35].  In that study, ingesting 15 g and 30 g of 
whey protein isolate after a bout of resistance exercise restored muscle protein synthesis rates to resting, 
fasting rates observed during energy balance in a dose dependent manner. This proposal will extend these 
findings and determine if the observed muscle protein synthetic plateau after conventional resistance exercise 
[9] during energy balance persists during moderate energy deficit when EAA are ingested relative to body 
mass (0.10 g/kg versus 0.30 g/kg). Relative EAA intakes were chosen to minimize inter-subject variability and 
to provide a spectrum of responses to varying absolute EAA doses in order to delineate the breakpoint, and 
practical dose, at which skeletal muscle and whole-body anabolic responses are optimal [44]. This study will be 
the first to assess whole-body protein kinetics in response to consuming different doses of EAA and their 
associations with muscle protein synthesis during short-term energy deficit.   
 
The stable isotope infusion protocol will occur in the morning following an overnight fast (Figure 6).  Two 
intravenous catheters will be placed; one for the infusion of stable isotopes, and the other heated for serial 
“arterialized” blood sampling.  As previously described [45], a baseline blood sample will be drawn (0-min) prior 
to primed continuous infusions of L-[ring-2H5]-phenylalanine and L-[3,3-2H2]-tyrosine. To appropriately reach 
isotopic equilibrium of L-[ring-2H4] tyrosine enrichment derived from L-[ring-2H5]-phenylalanine tracer, a priming 
dose of L-[ring-2H4]-tyrosine also will be injected at 0-min [45].  All isotopes will be purchased from Cambridge 
Isotope Laboratories (CIL, Andover, MA) and solutions will be constituted by a licensed pharmacy and certified 
sterile and pyrogen free before administration (pharmacy to be determined).  
 
At 120-min, a muscle biopsy of the vastus lateralis will be taken on the non-exercised leg only to assess basal 
(fasting/resting) muscle protein kinetics. After the muscle biopsy, volunteers will remain rested until they 
perform a heavy-load, high volume, single leg-resistance exercise bout at 195-min that consists of 8 sets X 10 
repetitions on the leg-press and leg-extension machines [9].  Both exercises will be performed at 80% of a pre-
determined one repetition maximum (1RM) with a 2-min rest interval between sets. Volunteers will have ~45-
min to complete the exercise bout. In the event the volunteer is unable to complete the prescribed workload; 
the workload will be reduced by 4.5 kg as previously described [9].  This will be repeated until the participant 
can complete the prescribed work. Approximately 5-min after completing the exercise bout (240-min), a muscle 
biopsy will be taken from the vastus lateralis of the exercised and non-exercised (rested) legs (the exercised 
and non-exercised leg will be the same between trials).  Immediately after the biopsies, volunteers will 
consume the appropriate dose (i.e., an artificially flavored beverage) of free-form EAA in 5-10-min 
[Reginator™; EAA content per 10 g of the mixture:  0.15 g histidine, 3.66 g leucine, 0.94 g isoleucine, 1.65 g 
lysine, 0.3 g methionine, 0.63 g phenylalanine, 0.89 g threonine, 0.1 g valine, 0.006 g tryptophan, and 0.81 g 
arginine (conditionally essential under stress)].  The phenylalanine content of the beverage will be artificially 

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enriched with L-[ring-2H5]-phenylalanine to 4% to limit potential disturbances in isotopic steady-state.  
Volunteers will remain rested during serial blood sampling over a 3-h period following ingestion.  Final biopsies 
will be taken from each leg at the end of the 3-h period.  There will be a total of 2 incisions per EAA trial, one 
on the rested leg and one on the exercised leg.  A total of 5 muscle biopsies will be collected from each 
volunteer per EAA trial; 2 through 1 incision on the exercised leg and 3 through 1 incision on the rested leg (2 
new incisions will be made on the subsequent EAA trial.  The incisions on EAA trial 1 will NOT be used for trial 
2).  Blood samples will be collected at regular intervals to assess isotopic enrichments for kinetic calculations, 
insulin, blood urea nitrogen, and amino acid concentrations (Figure 6).  
 
3-d Run-In and 5-d Energy Deficit 
Volunteers will complete a baseline, pre-study 3-d diet record (Appendix A) and a 3-d activity log (Appendix 
B) according to instructions provided by study team dietitians.  The data will be analyzed using Food Processor 
SQLTM (Salem, OR Version 10.0) and collected to predict volunteers’ total daily energy requirements for the 3-
d eucaloric run-in and 5-d energy deficit diets.  The volunteers will receive standardized meals derived 
primarily from military combat rations (Meals Ready-to-Eat, MRE) for each diet phase to replicate the source of 
nutrition during short-term military operations.  The energy content and macronutrient distribution of the MRE-
based, 3-d run-in diet will be sufficient to maintain body weight and provide between 50-65% carbohydrate and 
1.6 ± 0.2 g protein∙kg-1∙d-1, amounts habitually consumed by Warfighters in past USARIEM studies [39, 42]. 
Total energy content of the 5-d energy deficient diet will be reduced by 30%, primarily by reducing the total 
amounts of carbohydrate and fat consumed, while keeping dietary protein constant at 1.6 ± 0.2 g protein∙kg-1∙d-
1.  All meals will be prepared and provided to volunteers by study team dietitians during the 3-d run-in diet.  
During the 5-d energy deficit, all meals will be prepared and provided to the volunteers..  Volunteers will be 
completely restricted from all non-study physical activity during the 3-d run-in and 5-d energy deficit diets to 
minimize complications and the potential for carryover effects on study parameters.  If necessary, military 
volunteers will be provided study-specific profiles to restrict physical activity. This approach has been applied 
successfully in past USARIEM studies [39].  
 
During the 14-d washout period, volunteers will be instructed to consume an ad libitum diet.  Routine physical 
activity (exercise) can be resumed during the wash-out phase at levels consistent with amounts reported in 
pre-study 3-d activity records.  Diet records (24-h) and activity logs (24-h) will be completed every third day and 
then reviewed (the following day) during the 14-d washout period to assess dietary intake and physical activity. 
Volunteers will meet with study dietitians to review these records.  
 
Anthropometric Data 
Anthropometrics, performed using standardized techniques and equipment, will be used to characterize study 
volunteers, and evaluate responses to each 5-d energy deficit.  Height will be measured in duplicate to the 
nearest 0.1 cm using a stadiometer at baseline. Body weight will be measured, nude (scale will be placed in a 
locked bathroom) and after an overnight fast (≥ 8 h), using a calibrated digital scale to the nearest 0.1 kg at 
baseline and then daily during each 3-d run-in and 5-d energy deficit period to ensure weight maintenance 
and/or weight loss.  Body mass will be measured every third day during the 14-d washout period 
(measurements will correspond with the days diet records and activity logs are reviewed described above).   
 
Body composition will be determined at baseline and after the 14-d washout by using a dual energy x-ray 
absorptiometry (DEXA, DPX-IQ, GE Lunar Corporation, Madison, WI). The DEXA technique allows for the non-
invasive assessment of soft tissue composition by region with a precision of 1-3%. The volunteer will lay face-
up on the DEXA densitometer table in shorts, t-shirts, and stocking feet, and will be asked to remain 
motionless for the 8-10 min scan. These data will be used to calculate total body mass, fat-free mass, and fat 
mass. Calibration to external standards will be performed before actual data collection. The operator remains 
in the room with the volunteer during the scan. 
 
B5.4 Data Collection   
 
Blood Sampling and Total Blood Volume Collected 

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Serial blood draws collected during the stable isotope infusion studies (see Figure 6) will be analyzed for 
isotopic enrichments, blood urea nitrogen, insulin, and AA concentrations.  There are 16 blood draws per 
infusion study.  9.5mL of blood will be drawn at each of the 16 draws, for a total of 152 mL per infusion study 
and 304 mL over the course of the study.  Sufficient blood will be drawn and archived to allow for additional 
analyses of related metabolic biomarkers sensitive to EAA, resistance exercise, and energy deficit.   
 
Stable Isotope Analysis and Calculations of Whole-body and Muscle Protein Kinetics 
 
All stable isotope analyses on de-identified, coded blood and muscle samples will be conducted by Dr. 
Ferrando at UAMS (data sharing agreement attached is provided with this protocol). No samples will be stored 
at UAMS for future analyses because the entire sample provided will be processed for the assays below.  
 
Blood Sample Processing for Isotopic Enrichment  
Blood samples will be precipitated with 125 µL of 10% sulfosalicylic acid (SSA) and centrifuged. The internal 
standard technique will be used to determine blood EAA concentrations.  Free amino acids (AA) will be 
extracted from 300 µL supernatant fluid by cation exchange chromatography and dried under Speed Vac 
(Savant Instruments, Farmingdale, NY or similar model). Enrichments of phenylalanine and tyrosine will be 
measured on the tert-butyldimethylsilyl derivative with the use of gas chromatography-mass spectrometry 
(models 7890A/5975; Agilent Technologies, Santa Clara, CA or similar model) [46, 47]. Ions of mass-to-charge 
ratio of 234, 235, and 239 for phenylalanine, of 302 and 308 for leucine, and of 466, 467, 468, and 470 for 
tyrosine will be monitored with electron impact ionization and selective ion monitoring.  
 
Muscle Sample Processing for Isotopic Enrichment 
Muscle biopsy tissue samples taken from the vastus lateralis muscle will be cleaned of visible fat, blotted of 
excess blood, then frozen in liquid nitrogen, and stored in a −80°C freezer until later analysis. Upon thawing, 
muscle tissues will be weighed, and tissue proteins precipitated with 0.5 mL of 4% SSA. The tissues will then 
be homogenized and centrifuged for collection of supernatant. The procedure will be repeated two more times, 
and tissue intracellular free AAs extracted from the pooled supernatant via the same cation exchange 
chromatography stated in plasma analyses and then dried under the Speed Vac. The remaining muscle pellet 
will be washed, dried, and hydrolyzed in 0.5 mL of 6 N HCl at 105°C for 24-h. Enrichments from muscle free 
and bound tracers will be determined as in plasma analyses. 
 
Whole-body Protein Turnover and Muscle Fractional Synthesis Calculations 
Calculations of whole-body protein synthesis (PS) and protein breakdown (PB) rates will be performed based 
on the determinations of the rate of appearance (Ra) into the plasma of phenylalanine and tyrosine and the 
fractional Ra of endogenous tyrosine converted from phenylalanine [48]. Plasma enrichments of phenylalanine 
and tyrosine tracers will be curve-fitted with a cubic spline method using Graphpad Prism 5 (Graphpad 
Software, La Jolla, CA or equivalent). Whole body protein turnover will be calculated by dividing kinetic values 
of a single essential amino acid (i.e., phenylalanine), which cannot be synthesized in the body, by its fractional 
contribution to protein. For the calculations for whole-body PB rate, contribution from exogenous EAA and 
tracers infused will be subtracted from total Ra. Appropriate skew correction will be made for 13C-labeled 
tracers [48]. 
  
Calculation of resting and post-exercise muscle FSR will be determined as the incorporation of the 
phenylalanine tracer from biopsy one (180 min) to biopsy two (420 min) using the precursor-product model 
[49].  
  
The following equations will be used for calculations of whole-body and muscle protein kinetics: 
 
Total plasma Ra = F/E                                                             
Fractional Ra of Tyr from Phe = ETyr M+4/EPhe M+5                  
Phe hydroxylation = fractional Ra of Tyr from Phe X Ra|Tyr 

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PS = [(Ra|Phe –Phe hydroxylation) X 25]                                  
PB = [(Ra|Phe – FPhe) X 25 – PROMEAL]                                    
NB = PS – PB                                                                           
FSR (%) = [(Ep2-Ep1)/(Em X t)] X 60 X 100                   
 
where enrichment (E) is expressed as tracer-to-tracee ratio (TTR) for calculation of PB or mole percent excess 
for calculation of muscle FSR, calculated as TTR/(TTR + 1). E is enrichment of respective tracers at plateau. F 
is respective tracer infusion rate into a venous side: FPhe for phenylalanine tracer. ETyr M+4 and EPhe M+5 are 
plasma enrichments of tyrosine tracers at M+4 and M+5 relative to M+0, respectively. Correction factor of 25 is 
for conversion of value for phenylalanine to protein based on the assumption that contribution of phenylalanine 
to protein is 4% (100/4 = 25) [50]. PROMEAL is the amount of exogenous EAA (g) that appeared in the 
circulation, which was calculated as total amount of EAA provided (if any), based on the assumption that 85% 
of the EAA ingested was absorbed [51]. Phenylalanine hydroxylation is the Ra of tyrosine derived from 
phenylalanine through hydroxylation. EP1 and EP2 are the enrichments of bound L-[ring-2H5]-phenylalanine in 
the 180 min and 420 min biopsies, respectively. Em is the calculated mean value of the enrichments of [ring-
2H5]-phenylalanine in the intracellular or plasma pool. t is the time in minutes elapsed between muscle 
biopsies. Factors 60 and 100 were used to express muscle FSR in percent per hour. 
 
mRNA and microRNA Expression 
Total RNA will be isolated from approximately 25 mg of muscle using a mirVanaTM miRNA isolation kit 
(Invitrogen, Carlsbad, CA, USA) or equivalent.  Quantity and quality of RNA will be assessed using a Nanodrop 
ND-1000spectrophotometer (Nanodrop, Wilmington, DE, USA) or equivalent.  Equal amounts of total RNA will 
be synthesized into cDNA for analysis of mRNA (High-Capacity cDNA RT Kit, Applied Biosystems, Foster City, 
CA, USA or equivalent) and a TaqMan® microRNA RT kit (Applied Biosystems) or equivalent.  Individual 
primers will be used to determine the mRNA expression of known intracellular targets regulating protein 
metabolism, to include but not limited to atrogin, MurF-1. LARS, LAT1, SNAT2, REDD1, and IGF1.  Individual 
primers will be used to determine microRNA expression of microRNA that have been reported to impact 
skeletal muscle anabolism, to include but not limited to miR-1, miR-133a/b, miR-206, miR-208, miR-486, and 
miR-499 
 
To determine if circulating microRNA reflect changes within skeletal muscle to exercise and dietary protein 
intake total circulating microRNA will be extracted from 200 µL serum using miRNeasy Serum/Plasma kit or 
equivalent, which allows for extraction and purification of small (< 200 nt) cell-free RNA (Qiagen, Valencia, CA, 
USA).  Additionally, to assess if changes in circulating microRNA differ between total and exosomal circulating 
microRNA, exosomes will be extracted from 1000 µL serum using a miRCURYTM Exosome Isolation Kit-Serum 
and Plasma (Exiqon, Woburn, MA) or equivalents.  To avoid introduction of potentially contaminating material, 
prior to RNA extraction serum samples will be centrifuged for 10 min at 4°C to remove cellular debris.  
Supernatant will be removed and transferred to a new tube without disturbing the pellet.  Due to the small 
amount of RNA in the serum, 3.5 µL of a Spike-In Control (C. elegans miR-39; Qiagen) will be added to all 
samples prior to extraction of RNA to determine the yield of template recovered.  After extraction 3 μl of serum 
RNA will reverse transcribed using the TaqMan® microRNA RT kit (Applied Biosystems) or equivalent with 
miRNA-specific stem-loop RT primers pooled in 1X-Tris-EDTA (TE) buffer for a final dilution of 0.05X.  A pre-
amplification step will be performed after reverse transcription to increase cDNA template using a primer pool 
of 20 X Taqman® Small RNA Assays (Applied Biosystems) or equivalent for miRNA of interest at 0.05X 
concentration in 1X TE buffer.  All serum miRNA will be normalized to the geometric of external (Spike-In 
Control C. elegans miR-39) and internal controls to allow for both technical and inter-individual normalization. 
Geometric mean of controls will be used to correct for possible outlying values and abundance differences 
between the different controls [52].   
 
All reverse transcription for mRNA and miRNA, and pre-amplification of serum miRNA will be conducted in a 
T100TM Thermal Cycler (Bio-Rad, Hercules, CA or similar model). A StepOnePlusTM real-time PCR system 

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(Applied Biosystems or similar model) will be used to perform all mRNA and miRNA analysis.  Fold changes 
will be calculated using the ∆∆ cycle threshold (∆∆CT) method as described below in statistical analysis 
section. 
 
Western Blotting 
Approximately 30 mg of muscle will be homogenized in ice-cold buffer (1:10 wt/vol) containing 50 mM Tris–HCl 
(pH 7.5), 5 mM Na-pyrophosphate, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 10% glycerol (v/v), 1% Triton-X, 1 
mM DTT, 1 mM benz-amidine, 1 mM PMSF, 10 μg mL−1 trypsin inhibitor and 2 μg mL−1 aprotinin.  
Homogenate will be centrifuged for 15 min at 10,000 × g at 4°C.  Protein concentration of supernatant (lysate) 
will be determined using 660 nm Protein Assay (ThermoFisher Scientific, Waltham, MA, USA or equivalent).  
Phosphorylation status and total protein expression of molecular markers associated with anabolism will be 
determined using Western blotting techniques.  Muscle lysates will be solubilized in Laemmli buffer, with equal 
amounts of total protein (15 μg) separated by SDS-PAGE using precast Tris·HCl gels (Bio-Rad). Proteins will 
be transferred to polyvinylidene fluoride (PVDF) membranes and exposed to commercially available primary 
antibodies of intracellular markers involved with skeletal muscle anabolism and proteolysis to include but not 
limited to Akt, p-AktSer473, mTOR, p- mTORThr2448, p70S6K, p-p70S6KThr424, rpS6, and rpS6Ser235/236 (Cell 
Signaling Technology, Danvers, MA, USA or equivalent) at 4°C overnight.  Labeling will be performed using 
secondary antibody (anti-rabbit IgG conjugate with horseradish peroxidase; Cell Signaling Technology), and 
chemiluminescent reagent will be applied (Super Signal, West Pico Kit; Pierce Biotechnology, Rockford, IL, 
USA). Blots will be quantified using a phosphoimager (ChemiDoc XRS; Bio-Rad) and Image Lab software (Bio-
Rad) or similar models. To confirm equal protein loading per well a normalizing protein such as GAPDH or 
HSP90 will be assessed.  
 
B5.5 Managing Data and/or Human Biological Specimens for this Research  
 
All data and medical information obtained will be considered privileged and held in confidence.  Study 
volunteers will be assigned unique subject identification (ID) numbers that will not contain any personal 
identifiers such as name, social security number, address, date of birth, zip code, etc.  This study subject ID 
number will be used on all data collection instruments, to include questionnaires, data collection forms, 
computer records, etc. (Appendices A - D).  A number will be assigned as each volunteer is enrolled for 
participation.  A master list linking the volunteers’ names and ID numbers will be kept in a separate locked file 
in the principal investigator's or the project coordinator’s office, or kept in a computer file with password-
protected access restricted to the principal investigator and the project coordinator. Social security numbers 
and banking information will be collected to process volunteer payments.  The master list link, social security 
numbers, and banking information will be deleted immediately after the study has been completed and 
payment has been confirmed.  When the results of the research are published or discussed in conferences, no 
information will be included that would reveal identity.  Study samples will be processed on site at USARIEM 
and stored in Military Nutrition laboratory freezers (room 322, 304) usingthe subject identification number 
indefinitely for renalysis if necessary. Coded study samples (blood and muscle) for isotopic and amino acid 
analysis will be shipped on dry ice  to Dr. Arny Ferrando at UAMS via FedEx and stored in his laboratory until 
analyized.  Once analyzed, there will be no remaining sample for storage.  . 
 
Only personnel assigned to the research study by the principal investigator will have access to the data. Only 
the principal investigator and project coordinator will have access to personal identifiable data.  Hard copy data 
records will be stored for a minimum of three years from the time the study is completed.  Electronic data 
records will be maintained for a period of at least ten years after the study has been completed.   
 
B5.6 Managing Data and/or Human Biological Specimens for Future Research   
 
Any use of the samples outside of this defined protocol will be submitted as a protocol amendment or a new 
protocol.  Samples will be retained for further analyses related to metabolic biomarkers sensitive to EAA, 
resistance exercise, and energy deficit once the protocol is closed.  Participants will be made aware of this 
during the informed consent process.      

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B5.7 Devices, Drugs, Dietary Supplements, Nutritional Supplements, And Biologics   
 
B5.7.1 Devices 
 
5.7.1.1 FDA-approved device being used in this research according to the approved 
labeling  
DEXA, DPX-IQ, Lunar Corporation, Madison, WI 
 
 
5.7.1.2 FDA-approved device being used in this research in a manner other than its 
approved labeling 
N/A 
 
B5.7.2 Drugs 
                              
B5.7.2.1 FDA-approved and used in accordance with the approved labeling 
N/A 
 
B5.7.2.2 FDA-approved and used in a manner not in accordance with its approved 
labeling 
N/A 
 
B5.7.2.3 Any drug not approved by the FDA 
N/A 
 
B5.8 Statistical Analysis 
 
 
B5.8.1 Sample Size Estimation 
 
Means (SD) from Macnaughton et al. [53] and Witard et al. [9] demonstrating the effects of graded 
protein dose on resting and post-resistance exercise muscle FSR were used to determine statistical 
power and sample size.  Using those data, we anticipate observing a mean (SD) difference in muscle 
FSR of ~0.015 %/h (0.02 %/h) for both the rested and exercised leg between EAA doses.  The sample 
size necessary to determine differences between EAA doses (assuming an alpha of 0.05, mean 
difference of 0.015 %/h, standard deviation of 0.02 %/h, and 80% power) is 16.  However, 20 
volunteers will be studied to account for the potential for greater variability than those reported by 
others and to increase our statistical power to 90% in order to detect differences between the 0.10 g/kg 
and 0.3 g/kg EAA doses. This estimate also provides greater than 95% power to detect differences in 
whole-body protein balance between EAA doses [16].  We request the ability to increase our enrollment 
to 40 volunteers to account for screening failures and study attrition.  Enrollment and testing will cease 
once data has been collected on 20 volunteers.   
 
B5.8.2 Data analysis  
 
Statistical analyses will be conducted using either SPSS (IBM Corp. Armonk, NY), SAS 9.3 (SAS 
Institute Inc., Carey, NC), or equivalent.  Common descriptive statistics will be used to describe 
volunteer characteristics.  Shapiro-Wilk tests will be used to determine normality of data.  An ANOVA 
will be performed to determine the effects of EAA dose (0.10 g/kg vs. 0.3 g/kg) on muscle FSR and 
whole-body protein turnover.  Correlation coefficients and multiple regression analysis will be used to 
evaluate relationships between study outcome measures.  The alpha level will be adjusted for multiple 
comparisons, with the level for statistical significance set at P < 0.05.   
 

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SECTION C:  HUMAN RESEARCH PROTECTIONS 
 
C1.  RECRUITMENT AND CONSENT   
 
C1.1 Identification and Selection of Subjects  
 
Interested volunteers who have been briefed on study procedures will be provided the opportunity to 
consent to participate.  After consent and before medical clearance, study eligibility will be determined 
based on volunteer responses to questions pertaining to self-reported study inclusion and exclusion 
criteria (Appendix C). If still eligible, volunteers will make an appointment for a medical screening. If an 
individual fails the medical screening for whatever reason or does not meet eligibility criteria, their 
screening data will be destroyed.  
  
The medical clearance will take place at USARIEM (Natick, MA) by OMSO staff.  Volunteers recruited 
through SSIT may undergo medical clearance at their home duty station prior to arrival at USARIEM 
(clearances will be coordinated between the PI, OMSO, and the units Brigade Surgeon).The clearance 
will include a blood draw to assess health status and inclusion/exclusion criteria. The medical screening 
visit will take approximately 1 hr. If any medical screening tests show a possible medical concern, the 
volunteer will be notified. Those who receive study clearance and meet the inclusion/exclusion criteria 
will continue on to pre-study, baseline testing.   
 
C1.2 Recruitment Process   
 
Volunteers will be recruited from the federally and non-federally employed civilian population, the Natick 
Human Research Volunteer (HRV) Pool, NSSC active duty military personnel, and the active duty 
population located at other military organizations, to include coordination with NSSC Soldier/Squad 
Interface Team (SSIT).  
 
For HRVs, the Principal Investigator will provide a copy of the informed consent document to the 
Human Research Subjects Program Coordinator or designee. The Coordinator will schedule the 
consent briefing for the military human research volunteer platoon and will serve as ombudsman during 
the briefing. The HRV Coordinator may also organize consent briefings for Soldiers at their Advanced 
Individual Training unit.  The Coordinator will serve as an ombudsman for the offsite consent briefings.  
In addition, other military organizations may be recruited through coordination with NSSC SSIT.  The 
NSSC SSIT Coordinator will schedule the consent briefing for the military research volunteers and an 
ombudsman (either Katelyn Guerriere, Katherine Mitchell, Karleigh Bradbury, Maria Canino, Matthew 
Bartlett, or Caitlin Haven) will be present during the briefing.  
 
Superiors of Service members (e.g., unit officers, senior NCOs, and equivalent civilians) shall not be 
present at any recruitment sessions or during the consent process in which members of units under 
their command are afforded the opportunity to participate as human subjects of research. 
 
Civilian volunteers and other active duty personnel will be recruited by “word of mouth”, posted flyers 
(“off-site recruitment flyer” to be used for any flyers posted outside of NSSC, “recruitment flyer” to be 
used for postings within NSSC), or electronic distribution of the “off-site” flyer to include a text-only, 
approved version of the flyer.  Recruiting materials will be distributed around NSSC, surrounding 
community, and on bulletin boards at local universities. The text-based flyer will be posted on various 

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USARIEM social media sites and used in distribution media requiring a text format (e.g., electronic 
newsletters).  Recruiting may also be conducted through information meetings presented to college 
classes, clubs, sports teams, or other organizations. Approvals from the requisite parties will be 
obtained prior to any recruitment activities.  
 
C1.3 Eligibility   
 
All potential volunteers will complete the background questionnaire pertaining to the study inclusion and 
exclusion criteria (Appendix C). Volunteers must then be medically cleared by OMSO for participation 
in accordance with USARIEM procedures outlined for screening volunteers for research involving 
exercise.  Volunteers recruited through SSIT may undergo medical clearance at their home duty station 
prior to arrival at USARIEM (clearances will be coordinated between the PI, OMSO, and the units 
Brigade Surgeon).  Potential military and civilian volunteers will undergo the same clearances. 
Volunteers will be screened for anemia and problems with blood clotting, including prothrombin time 
(PT)/ partial thromboplastin time (PTT), which is a specific criterion for research involving muscle 
biopsies. Health problems identified during the screening process will be documented and a copy 
provided to the volunteer. The volunteer will be encouraged to make an appointment with their primary 
care provider for a full evaluation of the problem. Volunteers with evidence of any physical, mental, 
and/or medical conditions, as determined by OMSO, that would make the proposed studies relatively 
more hazardous will be excluded. Any personal health information collected during this screening 
process will be destroyed at the time of study withdrawal or at the completion of the study. 
 
All females will be given a urine pregnancy test during the initial screening and the morning of or at 
least 24 hours prior to each DEXA scan. The test will be read by a female member of the study team 
and the results will be shared with the volunteer. If the pregnancy test is positive, the volunteer will be 
excluded from further participation in this study.  
 
All volunteers must be willing to consume only food and beverages provided by study staff during the 
run-in and energy deficit phases of the protocol, and they must be willing to adhere to exercise and 
physical activity prescriptions and restrictions.  If the results of all screening tools reveal the volunteer 
fits the screening criteria, they will be eligible to volunteer for the study.   
 
C1.4 Consent Process 
 
Prior to providing informed consent, discussions with potential volunteers (such as over the telephone) 
will not involve the collection of any personally identifiable information besides their name, email, and 
telephone number. No study procedures will occur prior to any volunteer giving their informed consent. 
The principal investigator, an associate investigator or the project coordinator will brief potential 
volunteers about the nature, purpose, procedures involved, risks, expectations and requirements for 
participation in the study. Prospective volunteers will be familiarized with the study procedures and 
informed verbally and in writing of their rights to withdraw from any part of the study without penalty or 
prejudice. The principal investigator or designee will answer all group and private questions. Potential 
volunteers will have at least one hour after they are briefed, with the ombudsman remaining present, to 
read and review the Informed Consent document and decide whether they wish to consent to 
participate. An ombudsman will not be required for any individual briefings or for civilian group briefings. 
A copy of the informed consent document will be provided to the volunteer with the original kept for 
study documentation. If they meet all the medical selection and eligibility criteria after completing the 
screening health assessment and consenting to participate, they will begin preliminary testing. 
Volunteers who have already consented will be informed of any new information or changes to the 
protocol that may affect their willingness and ability to continue participation in the study using an 
approved consent addendum. 
 

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C1.4.1 Research involving subjects with cognitive impairment or who lack capacity to 
provide informed consent 
N/A 
 
C1.4.2 Research involving non-English speaking subjects 
N/A 
 
C1.4.3 Research involving a waiver of the requirement to obtain informed consent OR 
alteration of the elements of informed consent 
N/A 
 
C1.4.4 Research involving a waiver of the requirement for investigator to obtain a signed 
consent form 
N/A 
 
C1.4.5 Waivers of assent or parental permission when the research involves children 
N/A 
 
C1.4.6 Research involving data collection for the USAMRMC Volunteer Registry Database 
It is the policy of USAMRMC that data sheets are to be completed on all volunteers participating 
in this research for entry into the U.S. Army Medical Research and Materiel Command 
Volunteer Registry Database.  The information to be entered into this confidential database 
includes name, address, social security number, study name, and dates.  The intent of the 
database is twofold: first, to readily answer questions concerning an individual’s participation in 
research sponsored by the USAMRMC; and second, to ensure that the USAMRMC can 
exercise its obligation to ensure research volunteers are adequately warned (duty to warn) of 
risks and to provide new information as it becomes available.  The information will be stored at 
the USAMRMC for a minimum of 75 years. 
 
C2. COMPENSATION FOR PARTICIPATION   
 
Volunteers who participate in the study will receive $33.75 per blood draw (32 total draws), for a total of $1,080 
for completing the study.  
 
Note: Volunteers who receive more than $600 in a calendar year will have this income reported to the Internal 
Revenue Service.   
 
C3. WITHDRAWAL FROM RESEARCH PARTICIPATION  
 
Volunteers will be allowed to withdraw at any time without penalty or loss of benefits to which they would 
otherwise be entitled. An investigator may stop an individual’s participation in the study if the volunteer is 
unwilling or unable to complete study procedures. An investigator may also withdraw a volunteer if the 
individual becomes ill or injured or it would not be in the volunteer’s best interest to continue.  If the participant 
is withdrawn by the investigator or decides to voluntarily withdraw him/herself, all further data collection will 
discontinue.  Participants will be compensated for any blood draws they completed up until that point, and they 
will be asked to return any remaining food items that were provided, in addition to any wrappers and 
diet/activity logs that they had completed up to the point of withdrawal.  
 
C4. PRIVACY FOR SUBJECTS   
 
To protect the volunteer’s privacy, all of their research-related records will be labeled or “coded” with an 
assigned research volunteer number that will not include their name or any other form of identifiable 
information.  The principal investigator or project coordinator will keep the link between volunteer number and 

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the volunteer’s research records in a locked cabinet.  Any documents that will require the volunteer’s name, 
such as the consent form, will be kept in a locked cabinet separate from any research documents that contain 
the volunteer’s ID number.  The principal investigator and project coordinator are the only people who will be 
able to match the research volunteer number with any of their personal identifying information.    
 
When the results of the research are published or discussed in conferences, no information will be included 
that would reveal the volunteer’s identity to others.  If photographs, videos, or audio-tape recordings of 
volunteers are used for educational purposes, volunteer identity will be protected or disguised.  All identifiable 
or recognizable information (e.g., names and faces) will be covered in any photographs unless volunteers 
agree to sign a photo release form.  If volunteers do not sign a photo release form, any photographs taken of 
them will be destroyed. 
 
C5.  CONFIDENTIALITY PROCEDURES FOR RESEARCH RECORDS, DATA, HUMAN BIOLOGICAL 
SPECIMENS  
 
All data and medical information obtained will be considered privileged and held in confidence.  Study 
volunteers will be assigned unique subject identification (ID) numbers that will not contain any personal 
identifiers such as name, social security number, address, date of birth, zip code, etc.  This study subject ID 
number will be used on all data collection instruments, to include questionnaires, data collection forms, 
computer records, etc.  A number will be assigned as each volunteer is medically cleared for participation.  A 
master list linking the volunteers’ names and ID numbers will be kept in a separate locked file in the principal 
investigator's or project coordinator’s office, or kept in a computer file with password-protected access 
restricted to the principal investigator and project coordinator.  When the results of the research are published 
or discussed in conferences, no information will be included that would reveal identity.  Study samples will be 
processed and stored on site at USARIEM in Military Nutrition laboratories (rooms 304, 322).  Study samples 
will shipped to Dr. Arny Ferrando’s laboratory at UAMS will be processed in their entirety for analyses (i.e., no 
additional samples will be stored at UAMS).  All samples coded with a subject identification number.  The 
volunteers name or other identifiable information will not be included on any data, data collection sheets, 
specimens, or other research records.   
 
Only personnel assigned to the research study by the principal investigator will have access to the data.  Only 
the principal investigator and project coordinator will have access to personal identifiable data.  Hard copy data 
records will be stored for a minimum of three years from the time the study is completed.  Electronic data 
records will be maintained for a period of at least ten years after the study has been completed.   
 
C6.  RISKS OF HARM, MEASURES TO REDUCE THE RISKS OF HARM, AND BENEFITS OF 
PARTICIPATION 
 
C6.1 Risks of Harm  
     
Research Procedure Name:  Venous Catheterization 
Research Procedure Description:  A needle will be used to guide a catheter into a superficial vein. 
The catheter will either be attached to saline, or flushed periodically with saline, to keep the line patent 
for serial blood draws.     
Research-related Risks:  The risks of venous catheterization are small and usually limited to local 
bruising or swelling.  Sometimes volunteers feel faint or may faint during or right after the catheter is 
placed.  If the volunteer has had problems with fainting during blood draws in the past, they may be 
more prone to them during future procedures. Dizziness or faintness constitutes no long-term harm, 
and immediate relief is achieved by having the subject put their head down between their knees or lie 
down. If the catheter becomes clogged at any time during the protocol, it will be replaced to continue 
blood sampling and therefore the study. This will require another needle to be inserted.   

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Measures to Minimize Risks of Harm: Trained technicians will use aseptic techniques to place the 
catheter; however, in spite of being careful there is a chance that the site may become infected. 
Volunteers should not give blood for 8 weeks before or after this study.   
 
Research Procedure Name: Stable Isotope Administration 
Research Procedure Description: Volunteers will be infused intravenously with stable isotopes of 
amino acids on two occasions.   
Research-related Risks: The primary risks associated with tracer studies are those related to venous 
catheterization. The catheter can cause irritation, bruising, or infection.  There are no known risks or 
reported side effects associated with administration of stable isotopes to humans during clinical or 
experimental studies.  This is because there is relatively little mass difference between the isotopic 
tracers and the more prevalent natural isotopes, and the body’s naturally occurring pool of stable 
isotopes is high enough that types of experimental infusions proposed have no appreciable effect on 
the total abundance of the isotopes present in the body.  The risks associated with the infusion include 
volume overload, infection, and allergic reaction to the infused substance.  There have been no 
occurrences of volume overload, no occurrences of infection or allergic reaction attributable to iodine 
used prior to venipuncture in any of the 200 infusion protocols that the principal investigator has been 
involved with over the last 8 years.  To minimize the likelihood of these events occurring, infusion rate 
will be closely monitored and maintained at less than 50 ml/hr throughout the entire 7-h infusion period.  
Measures to Minimize Risks of Harm: All staff who directly participate in the stable isotope studies 
will be properly trained how to safely monitor (i.e., infusion pumps and IV lines) isotope infusion studies 
from Dr. Pasiakos, who has extensive experience with stable isotopes. In addition, infusates will be 
prepared sterile, pyrogen-free, and in the proper dosages by a licensed pharmacist and administered 
by Dr. Pasiakos, Dr. Margolis, and Dr. Gwin.   
 
Research Procedure Name:  Percutaneous Skeletal Muscle Biopsy 
Research Procedure Description: A small incision will be made in the skin and fascia of the vastus 
lateralis.  A 5-mm Bergstrom biopsy needle will pass through these incisions with manual suction 
applied to collect muscle samples, while the volunteer is under local anesthesia (1% lidocaine). 
Research-related Risks:  Percutaneous needle muscle biopsies have been established as a non-
routine, but safe research procedure. Similar to blood draws, there is a risk that volunteers will feel faint 
or may faint right after a muscle biopsy.  If the volunteer has had problems with fainting during blood 
draws or muscle biopsies in the past, they may be more prone to them during future procedures.  The 
most common risks associated with muscle biopsies are pain (~1.27%), erythema (~1.27%), and 
ecchymosis (1.27%) [54, 55]. Panic episode, bleeding, and edema have also been reported (0.21%, 
0.42%, and 0.84%, respectively) [54]. Denervation, numbness, and atrophy may occur but have not 
been verified in the literature.  Some minimal scarring will accompany healing of the incision and 
formation of a hypertrophic scar or keloid is possible.  Although this is a rare event in fair-skinned 
persons, the incidence of hypertrophic scarring or keloid formation associated with healing of a 
primarily closed skin biopsy site (i.e., one which was closed with sutures immediately afterward) is 5-
10% in dark-skinned persons.   
Measures to Minimize Risks of Harm: Complications of bleeding can be reduced by applying direct 
pressure to the wound following the biopsy.  If symptoms should occur, they usually do not interfere 
with normal walking or heavier exercise.  Volunteers with evidence of bleeding diathesis should be 
excluded during medical clearance; those with local skin infection or irritation or recent use of 
anticoagulant medication not identified during initial medical screening (including aspirin) will be 
withdrawn by the PI in consultation with OMSO. Volunteers will be instructed about precautions against 
hematoma and infection.  They will be given a handout outlining instructions for proper care of the 
incision site (Appendix D).  Muscle biopsies will be performed using sterile procedures by Dr. 
Pasiakos, Dr. Margolis, or Dr. Gwin, who will abide by USARIEM’s Percutaneous Skeletal Muscle 
Biopsy SOP (OMSO-approved USARIEM SOP for Invasive Procedures, Chapter 10) of 11 July 2017 in 
all regards. The PI and OMSO will follow-up with volunteers within 3 d post-biopsy to monitor for any 
sign of infection, bleeding, or hematoma. 

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Research Procedure Name:  Lidocaine Injection 
Research Procedure Description:  Approximately 8-10 mL of 1% lidocaine will be injected using a 25 
g needle at the site of the incision, superficially (i.e., skin) and within the vastus lateralis. 
Research-related Risks:  Slight pain at the site of injection might occur.  Although rare, anaphylactic 
reactions may also occur following administration of lidocaine.  Unlikely, but possible side effects could 
include: dizziness, confusion, shakiness, visual changes, nausea, and unusually slow heartbeat.   
Measures to Minimize Risks of Harm:  Volunteers will be instructed to notify a study investigator or 
the project coordinator immediately if an allergic (i.e., swelling, itching, rash, hives, difficulty swallowing, 
or difficulty breathing) reaction occurs. In the case of severe reaction, OMSO will be notified 
immediately and lidocaine use will be discontinued.  Dr. Pasiakos, Dr. Margolis, and Dr. Gwin will be 
the only ones administering the lidocaine, and medical staff will be onsite.  The PI and study staff will 
closely monitor the volunteers throughout the procedure.  
 
Research Procedure Name:  Resistance Exercise 
Research Procedure Description:  Resistance exercise includes maximal leg press and leg extension 
testing, and prescribed resistance exercise bouts during the infusion studies.   
Research-related Risks:  Resistance exercise is generally considered safe and the primary risks 
associated with resistance exercise include local muscle discomfort/soreness, ranging in intensity from 
mild to severe that can persist for 1-7 d.  Other risks include muscle strains and hyper-extension and/or 
flexion of the joint.    
Measures to Minimize Risks of Harm:  To reduce these risks, volunteers will be familiarized with the 
exercise tests and protocol well in advance of the criterion measure.  Volunteers will be closely 
monitored by experienced and qualified research team members, and asked to report any pain or 
discomfort resulting from exercise, followed up if necessary by medical examination and postponement 
or curtailment of further testing.  
 
Research Procedure Name:  Diet Interventions 
Research Procedure Description:  Volunteers will be instructed to follow a eucaloric 3-d run-in diet to 
maintain body mass, followed by a 5-d period of moderate energy deficit (20%).  Combat rations will be 
the primary food source in addition to some commercially available pre-packaged foods.     
Research-related Risks:  The foods and MRE components used in this study pose no known risks to 
volunteers. Sudden changes to the diet can cause gas, cramping, bloating, constipation, or other 
abdominal discomfort in some individuals. The main discomfort associated with a low energy diet is 
hunger. Volunteers will be shown copies of study menus and food lists at the initial study recruitment 
brief. This will be used to determine if prospective volunteers have an allergy, intolerance, or personal 
preference to foods listed. 
Measures to Minimize Risks of Harm:  All efforts will be made to accommodate the volunteers with 
regard to dietary preferences while keeping the major constituents of the diets consistent with study 
design and between volunteers. Those who have an allergy or intolerance to a menu component, which 
cannot be accommodated, will not be enrolled in the study. 
 
Research Procedure Name:  Dual energy X-ray absorptiometry (DEXA) scan 
Research Procedure Description:  Volunteers will lay face-up on the DEXA densitometer table in 
shorts, t-shirts, and stocking feet.  Volunteers will be asked to remain motionless for the 8-10 min scan. 
Research-related Risks:  The DEXA scan is an X-ray and is considered to be a low risk procedure.  
The radiation dose of the whole-body DEXA scan is 0.1 mrem.  This dose is equivalent to 
approximately 1/250 of normal annual background radiation, 1/9 of the radiation received in a 
transatlantic flight, or 1/30 of the radiation received in a chest X-ray.   
Measures to Minimize Risks of Harm:  A quality assurance check will be completed on the DEXA 
each day prior to its use; the software will not allow the use of the DEXA densitometer if the quality 
assurance check fails.  All females will be required to have a pregnancy test the day before, or the day 
of testing.  Pregnant females will not be scanned, nor allowed to continue the study. 

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C6.2 Incidental or Unexpected Findings   
 
Health problems identified during the screening process will be documented and a copy provided to the 
volunteer. The volunteer will be encouraged to make an appointment with their primary care provider 
for a full evaluation of the problem. Volunteers with evidence of any physical, mental, and/or medical 
conditions that would make the proposed studies relatively more hazardous will be excluded. 
 
C6.3 Potential Benefits   
 
There is no direct health or other benefits related to participation in this study. 
 
C7.  DATA AND SAFETY MONITORING   
 
C7.1 Monitoring 
 
The PI will, with the assistance of Associate Investigators and project coordinator, continuously 
evaluate recruitment, the informed consent process, adverse events, and protocol adherence and 
deviations in order to identify unanticipated problems or risks to the volunteers associated with the 
research. The PI will ensure that the number of volunteers recruited for this study complies with the 
protocol.  The PI will submit a monthly summary of all adverse events to the Research Monitor to 
determine whether the number of adverse events is excessive for the risks outlined in the research 
protocol.  The PI and onsite physician or PA will discuss “discontinuation criteria” for individual 
volunteers as the study progresses, based on their observations of the volunteer during testing or non-
testing periods. Every morning, volunteers will be asked the following questions to evaluate their 
readiness to test. 
 
• How have you been feeling since the last test in our laboratory (below average, average, above 
average)? 
• Do you have any pain or symptoms to report that may affect our testing today (e.g., sinus 
congestion, fatigue, muscle soreness, fever)? 
• Did you perform any exercise or physical activities outside of study activities in the last 24-h? 
 
C7.2 Research Monitor 
 
The research monitor for this study is MAJ Robin Cushing.  This individual is an appropriate subject 
matter expert not associated with the protocol. The research monitor shall, at a minimum, review all 
unanticipated problems involving risk to subjects or others, serious adverse events and all subject 
deaths associated with the protocol and provide an unbiased written report of the event.  Other 
responsibilities may be assigned by the USA MRMC IRB as needed. 
 
C8.  REPORTABLE EVENTS    
 
C8.1 Expected adverse events 
 
An adverse event is defined as any untoward or unfavorable medical occurrence in a human research 
volunteer, including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, 
or disease, temporally associated with the individual’s participation in the research, whether or not 
considered related to the individual’s participation in the research. 
 
A Serious Adverse Event is defined as any adverse event temporally associated with the subject’s 
participation in research that is fatal, life-threatening, permanently disabling, requires inpatient 
hospitalization, or results in congenital anomalies/birth defect, overdose or cancer, or based on 

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appropriate medical judgment, may jeopardize the volunteer, or may require medical or surgical 
intervention to prevent one of the above outcomes. 
 
 
All medical events that the USARIEM Office of Medical Support and Oversight (OMSO) evaluates will 
be reported to the ORQC.  The PI will report all adverse events to the research monitor, if one was 
appointed for the study.   
 
Expected adverse events which are not serious are reported to the IRB at the time of continuing review 
of the protocol.  These events include bruising, infection, swelling and slight pain from the IV 
placement; slight pain from the lidocaine injection; pain, soreness, infection, bruising from the muscle 
biopsy; feeling faint with IV placement, blood draw or biopsy; fatigue and muscle soreness from study 
exercises; hunger, bloating, gas, cramping, constipation from the dietary invention; fatigue and 
headaches during the negative energy balance portion of the dietary intervention. 
 
C8.2 Unexpected adverse events and unanticipated problems 
 
All unanticipated problems involving risk to subjects or others, and serious adverse events that are 
unexpected and determined to be at least possibly or definitely related to study participation, will be 
promptly reported within one working day by phone (508-233-6306/4811) or email 
(usarmy.natick.medcom-usariem.mbx.usariem-rqc@mail.mil) to the USARIEM ORQC and the 
Commander.  These events will also be reported to the HQ USAMRMC Institutional Review Board 
within one working day by phone (301-619-6240), or by e-mail (usarmy.detrick.medcom-
usamrmc.other.irb-office@mail.mil). 
 
Adverse events assessed by the PI as not serious and serious adverse events that are deemed to be 
unrelated to participation in the study will be reported to the IRB at the time of continuing review of the 
protocol. 
 
The research monitor is required to review all unanticipated problems involving risk to volunteers or 
others, serious adverse events and all volunteer deaths associated with the protocol and provide an 
unbiased written report of the event.  At a minimum, the research monitor should comment on the 
outcomes of the event or problem, and in the case of a serious adverse event or death, comment on 
the relationship to participation in the study.  The research monitor should also indicate whether he or 
she concurs with the details of the report provided by the study investigator.  Reports for events 
determined by either the investigator or research monitor to be possibly or definitely related to 
participation will be promptly forwarded to the ORQC and HQ USAMRMC IRB. 
 
C8.3 Adverse device effects: N/A 
 
C8.4  FDA-regulated research under IND and IDE: N/A 
 
 
 
SECTION D:  REFERENCES 
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Margolis, L.M., et al., Effects of winter military training on energy balance, whole-body protein balance, muscle 
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4. 
Volpi, E., et al., Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein 
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5. 
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Katsanos, C.S., et al., Aging is associated with diminished accretion of muscle proteins after the ingestion of a 
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Witard, O.C., et al., Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing 
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Pasiakos, S.M., et al., Efficacy and safety of protein supplements for U.S. Armed Forces personnel: consensus 
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Zhang, X., et al., An isotopic method for measurement of muscle protein fractional breakdown rate in vivo. Am J 
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Tuvdendorj, D., et al., Comparison of bolus injection and constant infusion methods for measuring muscle protein 
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Tuvdendorj, D., et al., A novel stable isotope tracer method to measure muscle protein fractional breakdown rate 
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Wolfe, R.R. and S.L. Miller, Amino acid availability controls muscle protein metabolism. Diabetes Nutr Metab, 
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Pasiakos, S.M., Exercise and amino acid anabolic cell signaling and the regulation of skeletal muscle mass. 
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Drummond, M.J., et al., An increase in essential amino acid availability upregulates amino acid transporter 
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Moberg, M., et al., Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more 
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Dreyer, H.C., et al., Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and 
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McCarthy, J.J., The MyomiR network in skeletal muscle plasticity. Exerc Sport Sci Rev, 2011. 39(3): p. 150-4. 
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Drummond, M.J., et al., Aging differentially affects human skeletal muscle microRNA expression at rest and after 
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30. 
Zacharewicz, E., et al., Identification of microRNAs linked to regulators of muscle protein synthesis and 
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31. 
Camera, D.M., et al., Selective Modulation of MicroRNA Expression with Protein Ingestion Following Concurrent 
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32. 
Margolis, L.M., et al., Skeletal Muscle myomiR Are Differentially Expressed by Endurance Exercise Mode and 
Combined Essential Amino Acid and Carbohydrate Supplementation. Front Physiol, 2017. 8: p. 182. 
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Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
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Date: 15 January 2019  
 
 
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SECTION E:  ABBREVIATIONS AND ACRONYMS 
 
AA, amino acids; EAA, essential amino acids; MRE, Meal Ready-to-Eat; NEAA, non-essential amino acids; 
FSR, fractional synthesis rate; FBR, fractional breakdown rate; PS, protein synthesis; PB, protein breakdown; 
net, net protein balance; DEXA, dual energy x-ray absorptiometry 
 
 
 
SECTION F: DoD PRIVACY RULE AND PROTECTED HEALTH INFORMATION (HIPAA) 
Click in the appropriate box See the “Guide for Investigators” for definitions and further information.   
 

Protocol Title: The effects of varying essential amino acid intakes on resting and post-exercise skeletal muscle and whole-body protein kinetics during 
negative energy balance 
Version # 5   
Date: 15 January 2019  
 
 
Protocol_Clinicaltrials.Gov 21OCT14 
 
Page 31 of 31 
 
 NA – institution is not a covered entity 
 
 NA – will not use or disclose protected health information 
 
 HIPAA authorization will be obtained  
 
 An application for waiver/alteration of HIPAA authorization will be submitted