The Mechanistic Effects of Acute Hypobaric Hypoxia on Exogenous Carbohydrate Utilization During Steady-state Aerobic Exercise
Phase: NA
Conditions: Glucose Metabolism, High Altitude
Sponsor: United States Army Research Institute of Environmental Medicine

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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PROTOCOL TITLE:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate 
1 
utilization during steady-state aerobic exercise   
2 
 
3 
SECTION A:  RESEARCH TEAM AND LOCATIONS 
4 
 
5 
A1.  RESEARCH TEAM 
6 
 
7 
Study Role 
Institution/Company and Contact Information 
Sponsor 
Organization/Institution/Company:  N/A 
 
Principal Investigator 
Name, Rank, and Degree: Lee M. Margolis, PhD, RD 
Title:  ORISE Post Doctoral Fellow 
Institution:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick MA 
01760 
Phone Number:  508-233-4591 
Email:  lee.m.margolis.ctr@mail.mil 
 
Associate Investigator(s) 
 
Name, Rank, and Degree:  Stefan M. Pasiakos, PhD 
Title:  Nutritional Physiologist 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-6474 
Email:  stefan.m.pasiakos.civ@mail.mil 
 
 
Name, Rank, and Degree:   Andrew J. Young, PhD 
Title: Nutritional Physiologist, ORISE Fellow 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-5141 
Email:  Andrew.j.young.ctr@mail.mil 
 
Name, Rank, and Degree:  Robert W. Kenefick, PhD 
Title:  Physiologist 
Institution/Company:  USARIEM / Thermal Mountain Medicine Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-6344 
Email:  robert.w.kenefick.civ@mail.mil 
 
Name, Rank, and Degree:  Arny A. Ferrando, PhD 
Title:  Professor 
Institution/Company:  University of Arkansas for Medical Science 
(UAMS) 
Address:  4301 West Markham St, Slot 806 
Phone Number:  501-526-5711 
Email:  AFerrando@uams.edu 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Name, Rank, and Degree:  Scott J. Montain, PhD 
Title:  Division Chief 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-4564 
Email:  scott.j.montain.civ@mail.mil 
 
Name, Rank, and Degree:  Roy M. Salgado, PhD 
Title:  Physiologist 
Institution/Company:  USARIEM / Thermal Mountain Medicine Dvision 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-5959 
Email:  roy.m.salgado.civ@mail.mil 
 
Name, Rank, and Degree:  Jessica A. Gwin, PhD 
Title:  ORISE Post Doctoral Fellow 
Institution/Company:  USARIEM / Military Nutrition Dvision 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-6492 
Email:  jessica.a.gwin.ctr@mail.mil 
Other Key Research  
Personnel (as applicable) 
 
Name, Rank, and Degree:  Marques Wilson, MS 
Title:  Project Coordinator 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233- 5681 
Email:  marques.a.wilson.civ@mail.mil 
 
Other Individuals 
Supporting  
the Research (as 
applicable) 
 
Name, Rank, and Degree:  Nancy Murphy, MS 
Title:  Laboratory Manager 
Institution/Company:  USARIEM / Military Nutrition Division 
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 Assistant 
Institution/Company:  USARIEM / Military Nutrition Division 
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:  Claire Whitney, MS, RD 
Title:  Research Dietitian 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-4875 
Email:  claire.c.whitney.civ@mail.mil 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Name, Rank, and Degree:  Adrienne Hatch, MS, RD 
Title:  Dietitian 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-5648 
Email:  adrienne.m.hatch.civ@mail.mil 
 
 
Name, Rank, and Degree:  Karleigh Bradbury, MS 
Title:  ORISE Research Fellow 
Institution/Company:  USARIEM / Thermal Mountain Medicine 
Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-4977 
Email:  Karleigh.e.bradbury.ctr@mail.mil 
 
Name, Rank, and Degree:  Katherine M. Mitchell, MS 
Title:  Research Technician 
Institution/Company:  USARIEM / Thermal Mountain Medicine 
Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-4177 
Email:  Katherine.m.mitchell15.civ@mail.mil 
 
 
Name, Rank, and Degree:  Beau R. Yurkevicius, BS 
Title:  ORISE Research Fellow 
Institution/Company:  USARIEM / Thermal Mountain Medicine 
Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-5950 
Email:  beau.r.yurkevicius.ctr@mail.mil 
 
 
Name, Rank, and Degree:  Pedro J. Claro, BS 
Title:  Chamber Crew Chief 
Institution/Company:  USARIEM / Thermal Mountain Medicine 
Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-5059 
Email:  pedro.j.claro.civ@mail.mil 
 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Name, Rank, and Degree:  SGT Ray Rivera 
Title:  Chamber Crew Chief 
Institution/Company:  USARIEM / Thermal Mountain Medicine 
Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-4041 
Email:  raymond.riverakuilan.mil@mail.mil 
 
Name, Rank, and Degree:  SGT Brandon Cordell 
Title:  Altitude Chamber Operator 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-6985 
Email:  brandon.j.cordell.mil@mail.mil 
 
 
 
Name, Rank, and Degree:  SGT Alfonzo Patino, BS 
Title:  Altitude Chamber Operator 
Institution/Company:  USARIEM / Military Nutrition Division 
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 Cassandra Rousayne 
Title:  Altitude Chamber Operator 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-5140 
Email:  cassandra.l.rousayne.mil@mail.mil 
Name, Rank, and Degree:  SPC Jason Alaniz 
Title:  Altitude Chamber Operator 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  N/A 
Email:  jason.alaniz3.mil@mail.mil 
Name, Rank, and Degree:  Patrick Radcliffe, BS 
Title:  ORISE Research Fellow 
Institution/Company:  USARIEM / Military Nutrition Division 
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:  Heather Fagnant, MS/MPH, RD 
Title:  ORISE Research Fellow 
Institution/Company:  USARIEM / Military Nutrition Division 
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 mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Name, Rank, and Degree:  Claudia Toussaint, MPH  
Title:  Chamber Crew Cheif 
Institution/Company:  USARIEM / Thermal Mountain Medicine 
Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508) 233-5956 
Email:  claudia.m.toussaint.ctr@mail.mil 
 
 
Name, Rank, and Degree:  LTC Frank Petrassi, PhD  
Title:  Chamber Crew Cheif 
Institution/Company:  USARIEM / Thermal Mountain Medicine 
Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508) 233-4861 
Email:  frank.petrassi.mil@mail.mil 
Research Monitor 
Name, Rank, and Degree:  MAJ Robin Cushing, DPHS 
Title:  Physician Assistant 
Institution/Company:  USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  253-241-6526 
Email:  robin.e.cushing.mil@mail.mil 
 
Ombudsman 
Name, Rank, and Degree:  Katelyn Guerriere, MS 
Title:  Ombudsman 
Institution/Company:  USARIEM / Military Performance Division 
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:  Sarah Ross 
Title:  Ombudsman 
Institution/Company:  Human Research Volunteer Program 
Address:  10 General Greene Ave, Bldg 2, Natick, MA 01760 
Phone Number:  (508)233-5619 
Email:  sarah.e.ross26.civ@mail.mil 
 
 
 
A2.  ROLES AND RESPONSIBILITIES 
1 
      
2 
       A2.1  Key Research Personnel   
3 
            Name(s):  Lee M Margolis 
4 
Research Role:  Principal Investigator  
5 
Study Responsibilities:  The Principal Investigator is responsible for the safe and scientifically sound 
6 
conduct of the study.  He will oversee all aspects of study, ensure safety and ethical treatment of 
7 
volunteers; maintain required documentation for the study and obtain required approvals; and will 
8 
have primary responsibility for data analysis, interpretation, and publication. Dr. Margolis will also 
9 
be actively involved in the volunteer brief, obtaining consent, data collection, performing muscle 
10 
biopsies, catheterization, phlebotomy, and exercise testing and interventions.   
11 
 
12 
Name(s):  Stefan M Pasiakos 
13 
Research Role:  Associate Investigator  
14 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, 
15 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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hypotheses, experimental approach and design. Assist PI with volunteer brief, obtain consent, 
1 
perform muscle biopsies, catheterization, phlebotomy, exercise testing and interventions, data 
2 
collection, management, and analysis and manuscript preparation.  
3 
 
4 
Name(s):  Andrew J Young, Robert W Kenefick, Scott J Montain, Roy M Salgado, and Jessica A 
5 
Gwin  
6 
Research Role:  Associate Investigators  
7 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, 
8 
hypotheses, experimental approach and design. Assist PI with data collection, management, and 
9 
analysis and manuscript preparation. 
10 
 
11 
Name(s):  Arny A Ferrando 
12 
Research Role:  Associate Investigator 
13 
Study Responsibilities:  Assist PI with the design, analysis, and interpretation of the stable isotope 
14 
studies of glucose metabolism.  Dr. Ferrando will receive and analyze coded, de-identified blood 
15 
samples for isotopic analysis.  He will not interact or intervene with study volunteers or have access 
16 
to personal identifiable information. A coded specimen transfer agreement is included with this 
17 
submission.  
18 
 
19 
            Name(s):  Marques Wilson 
20 
Research Role:  Project Coordinator  
21 
Study Responsibilities:  Supervise, manage, and coordinate study logistics and biological data 
22 
collection. He will be involved with study implementation. He will actively participate in data 
23 
collection to include catheterization, phlebotomy, exercise testing and interventions, and DEXA 
24 
scans.  He will assist in management, analysis and interpretation of data. 
25 
 
26 
       A2.2. Others Involved in the Research, as applicable 
27 
Name(s):  Claire Whitney and Adrienne Hatch 
28 
Research Role:  Study Dietitian 
29 
Study Responsibilities:  Baseline diet assessments, study diet development, and prepare and 
30 
administer test diets to volunteers.  She will actively participate in data collection to include exercise 
31 
testing and interventions, phlebotomy, and DEXA scans.  She will assist in management, analysis 
32 
and interpretation of data. 
33 
 
34 
Name(s):  Nancy Murphy  
35 
Research Role:  Biological Sample Coordinator  
36 
Study Responsibilities:  Supervision, management, and coordination of logistics and biological data 
37 
collection and analysis. She will be involved with protocol development, study implementation.  Data 
38 
collection responsibilities will involve sample processing. 
39 
 
40 
Name(s):  Christopher Carrigan   
41 
Research Role:  Research Assistant 
42 
Study Responsibilities:   Assist with data collection and biological sample processing and analysis. 
43 
Data collection responsibilities will involve with DEXA measurements, phlebotomy, and 
44 
catheterization. 
45 
 
46 
Name(s):  Karleigh Bradbury, and Beau Yurkevicius  
47 
            Research Role:  Research Technician 
48 
Study Responsibilities:  Assist with data collection to include catheterization, phlebotomy, exercise 
49 
testing and interventions, DEXA scans and biological sample processing and analysis.  
50 
 
51 
Name(s):  Katherine Mitchell  
52 
            Research Role:  Research Technician 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Study Responsibilities:  Assist with data collection to include exercise testing and interventions and 
1 
DEXA scans.  
2 
 
3 
Name(s):  Pedro Claro, SGT Ray Rivera, Claudia Toussaint, and LTC Frank Petrassi  
4 
Research Role:  Altitude Chamber Crew Chief 
5 
Study Responsibilities:  Will act as the Altitude Chamber Crew Chief, coordinate schedule for 
6 
altitude chamber, and supervise altitude chamber operation. 
7 
 
8 
Name(s):  SGT Brandon Cordell, SGT Cassandra Rousayne, SGT Alfonso Patino, and SPC Jason 
9 
Alaniz 
10 
Research Role:  Altitude Chamber Operator 
11 
Study Responsibilities:  Will act as the Altitude Chamber Operator. 
12 
 
13 
Name(s):  Patrick Radcliffe and Heather Fagnant  
14 
            Research Role:  Research Technician 
15 
Study Responsibilities:  Assist with diet preparation and data collection. 
16 
 
17 
Name(s):  MAJ Robin Cushing 
18 
Research Role:  Research Monitor 
19 
Study Responsibilities:  The research monitor for this study is MAJ Robin Cushing. MAJ Robin 
20 
Cushing is an appropriate subject matter expert not associated with the protocol. The research 
21 
monitor shall, at a minimum, review all unanticipated problems involving risk to subjects or others, 
22 
serious adverse events and all subject deaths associated with the protocol and provide an unbiased 
23 
written report of the event.  The PI and research monitor will discuss ‘discontinuation criteria’ for 
24 
individual volunteers as the study progresses, based on their observations of the volunteer during 
25 
testing or non-testing periods. 
26 
 
27 
Name(s):  Katelyn Guerriere and Sarah Ross 
28 
Research Role:  Ombudsman 
29 
Study Responsibilities:  Will act as the ombudsman for this study as necessary. 
30 
 
31 
A3.  RESEARCH LOCATIONS    
32 
 
33 
USARIEM, Natick MA: The U.S. Army Research Institute of Environmental Medicine (USARIEM) is a 
34 
DoD research facility within the U.S. Army Medical Research and Materiel Command.  It is the Institute 
35 
responsible for conducting basic and applied research to determine the effects of exposure to 
36 
environmental extremes, occupational tasks, physical training, deployment, operational stress and 
37 
nutritional factors on the health and performance of military personnel.  The facility contains 
38 
environmental chambers for controlling temperature and humidity, an environmentally controlled 
39 
hypobaric chamber, a water immersion laboratory, as well as several dry and wet laboratories for 
40 
animal and human experimentation.  The dry laboratories are capable of a broad range of experiments, 
41 
including biomechanical analysis, body composition, energy expenditure, muscle strength and 
42 
endurance.  The wet laboratories include general clinical chemistry analyzers, as well as equipment for 
43 
ELISA, RIA, histology, and molecular biology assays.  Each investigator at the facility has a personal 
44 
computer with software for data management, analysis, presentation and report generation.  Their 
45 
computers are interfaced with a network server for easy, secure data handling and transfer.  All testing 
46 
procedures will take place at USARIEM. 
47 
 
48 
University of Arkansas Medical Science, Center for Translational Research in Aging and Longevity, 
49 
Little Rock, AR: The Center for Translation Research in Aging & Longevity (CTRAL), Donald W 
50 
Reynolds Institute of Aging (RIOA), University of Arkansas for Medical Science (UAMS) has the basic 
51 
laboratory facilities and equipment to analyze stable isotope kinetics, including 2 Agilent 5973 GC/MS, 
52 
and 3 Agilent 5975 GC/MS, a Finnegan TSQ 7000 LC/MS/MS, and a Waters QTOF LC/MS.  The 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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CTRAL is led by the world’s foremost expert in stable isotope assessments of human metabolism, Dr. 
1 
Robert R Wolfe. 
2 
 
3 
Metabolic Solutions, Nashua, NH: Metabolic Solutions is a state-of-the art stable isotope analytical 
4 
laboratory that has the basic laboratory facilities and equipment to analyze stable isotope in breath and 
5 
blood.  Equipment onsite includes an Agilent 6110 LC-Tandem Mass Spectrometer (LC-MS/MS) Triple 
6 
Quad with Agilent 1100 LC, Thermo Finnigan Delta XP Isotope Ratio Mass Spectrometer (IRMS) with 
7 
GC Isolink for 13C, 15N and 2H GC-IRMS and Conflow IV interface, and a Thermo Finnigan Delta V 
8 
Advantage IRMS with trace GC-Combustion III unit for 13C, 15N and D GC-C-IRMS and Conflow IV 
9 
interface and elemental Analyzer EA112HT 13C, 15N and D sample combustion unit. Metabolic 
10 
Solutions has been an industry leader in developing new stable isotope tracer applications.  Metabolic 
11 
Solutions will analyze samples on a fee for service basis. 
12 
 
13 
Pennington Biomedical Research Center (PBRC), Baton Rouge, LA: The laboratory is accredited by 
14 
the College of American Pathologists (CAP) and participates in proficiency testing programs 
15 
administered by the CAP. Biochemical analyses will be conducted at PBRC on a fee for service basis.   
16 
 
17 
 
18 
 
19 
 
20 
SECTION B:  RESEARCH METHODOLOGY 
21 
 
22 
B1.  ABSTRACT   
23 
Service members deployed to high altitude (HA; hypobaric hypoxia) regions of the world experience 
24 
aerobic performance decrements. Factors contributing to hypoxia-associated aerobic performance 
25 
decrements, include an alteration in carbohydrate oxidation to metabolize energy to support the muscle 
26 
workload. However, research has failed to consistently demonstrate an ergogenic advantage of 
27 
consuming supplemental carbohydrate during strenuous work at HA.  Moreover, there is evidence that 
28 
oxidation of exogenous carbohydrate during exercise is blunted under acute hypobaric hypoxic 
29 
conditions compared to normoxia (1, 2).  The mechanisms by which hypoxia suppresses exogenous 
30 
carbohydrate oxidation are not known.  A better understanding how hypoxia modulates energy 
31 
substrate metabolism during exercise is necessary for appropriate design and implementation of 
32 
nutritional interventions aimed at maintaining physical performance at HA.  The primary objective of this 
33 
study is to confirm that acute HA exposure decreases exogenous carbohydrate oxidation during 
34 
steady-state aerobic exercise compared to SL, and explore if the mechanism inhibiting plasma glucose 
35 
uptake is insulin dependent or independent.  This randomized crossover study will examine substrate 
36 
metabolic responses to ingesting supplemental carbohydrate during steady-state aerobic-type exercise 
37 
at sea level (SL) and following acute (~5 h) exposure to HA (4,300 m) conditions in 10 healthy, 
38 
recreationally active males between the ages of 18-39 yrs.  Following a 48-hr muscle glycogen 
39 
normalization period, volunteers will complete 80-min of metabolically-matched, steady-state aerobic 
40 
(same absolute workload corresponding to ~55 ± 5 % of VO2peak at HA) exercise on a treadmill, and 
41 
consume 145 g of glucose (1.8 g·min-1) at SL and HA.  SL and HA trials will occur in the USARIEM 
42 
hypobaric chamber and be separated by a minimum 7-d washout period.  Glucose tracers will be used 
43 
to assess glucose turnover, and contribution to exogenous and plasma glucose oxidation.  Indirect 
44 
calorimetry and urine collections will be used to determine total carbohydrate, fat, and protein oxidation 
45 
during exercise at SL and HA.  Serial blood draws will be collected during each trial to assess 
46 
endocrine and circulating substrate responses to exercise, carbohydrate, and hypoxia. Muscle biopsies 
47 
will be collected before and after steady-state exercise to examine muscle glucose transporter 
48 
expression and translocation, glycogen status, and activity intramuscular enzymes in aerobic and 
49 
anaerobic energy metabolism.  The primary risks associated with this study include those associated 
50 
with acute hypobaric hypoxia, exercise, blood draws, and muscle biopsies.   
51 
 
52 
B2.  BACKGROUND AND SIGNIFICANCE   
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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1 
Ingesting carbohydrate during aerobic exercise conducted at SL increases exogenous carbohydrate 
2 
oxidation and limits endogenous carbohydrate utilization (3-6). Only two previous studies have 
3 
investigated how hypoxia affects oxidation of exogenous carbohydrate ingested during exercise, and 
4 
the observations reported from those studies differ (1, 2). When carbohydrate was ingested during 
5 
exercise at 77% V̇ O2max in normoxia and hypoxia equivalent to 4,300 m, exogenous carbohydrate 
6 
oxidation was the same (2).  In contrast, when carbohydrate was ingested during lower intensity 
7 
exercise (55% Wmax, ~55-60% V̇ O2max), exogenous carbohydrate oxidation was lower at high altitude 
8 
(HA; 3,375 m) than normoxia (1). The reasons for the discrepant effects of hypoxia observed in these 
9 
studies on exogenous carbohydrate oxidation during exercise are not clear, but might reflect 
10 
differences in the exercise intensities employed.  
11 
 
12 
Hypoxia reduces V̇ O2max (7, 8), so in the experiments described above (1, 2), exercise was performed 
13 
at lower absolute intensity and metabolic rate in hypoxia to maintain relative exercise intensity (i.e. % 
14 
V̇ O2max) the same as normoxia. However, from a scientific (9, 10) and practical perspective (the 
15 
metabolic rate during physical activities is the same at SL and 
16 
HA, notwithstanding differences in relative intensity (i.e., % 
17 
V̇ O2max).  We believe that differences in substrate metabolism 
18 
during exercise at HA and SL should be investigated at the 
19 
same absolute workload in both conditions. In one of the 
20 
aforementioned studies (2), Péronnet et al. also compared 
21 
exogenous carbohydrate oxidation during exercise performed at 
22 
the same absolute intensity and metabolic rate (i.e., V̇ O2 = ~ 2.2 
23 
L·min-1, 11.6 kca·min-1) in normoxia (54% V̇ O2max) and hypoxia 
24 
(78% V̇ O2max) equivalent to 4,300 m.  When subjects consumed 
25 
140 g of glucose during aerobic exercise that was metabolically 
26 
matched exogenous glucose oxidation was numerically lower 
27 
but not statistically different at HA compared to SL, while total 
28 
and endogenous carbohydrate oxidation were significantly increased at HA compared to SL. Maximal 
29 
exogenous carbohydrate oxidation rates are achieved between 51-64% of V̇ O2max (11). Therefore, in 
30 
this previous study (2), it is possible that exogenous carbohydrate oxidation was already maximal 
31 
during the normoxic exercise trials and could not increase any further during the hypoxic exercise.  In a 
32 
recent investigation, our laboratory sought to examine whether oxidation rates of exogenous glucose 
33 
would differ during exercise performed at the same absolute intensity in normoxic and hypoxic 
34 
conditions at exercise intensities eliciting less than maximal exogenous carbohydrate oxidation rates at 
35 
HA.  Findings from this study (unpublished) indicated that exogenous glucose oxidation diminished by 
36 
46% from SL to HA when participants consumed 144 g carbohydrate while walking on a treadmill for 
37 
80-min at a fixed absolute workload (V̇ O2 = ~1.7 L·min-1; 55% V̇ O2max at HA; Figure 1).  While 
38 
exogenous glucose oxidation was lowered in our investigations, endogenous carbohydrate oxidation 
39 
was numerically but not statistically higher at HA compared to SL.  These data suggested that the 
40 
body’s ability to oxidize exogenous glucose during exercise may be compromised during acute HA 
41 
exposure.    
42 
 
43 
Decreased insulin sensitivity is one potential mechanism for diminished ability to oxidize exogenous 
44 
carbohydrate oxidation at HA (12-15).  Concomitant with the suppression of exogenous carbohydrate 
45 
oxidation in acute hypoxia, we also observed that blood glucose levels rose higher and remained 
46 
elevated during exercise despite two to four fold higher insulin concentrations during HA exercise 
47 
compared to SL (Figure 2).  Under normal physiological conditions, elevated blood glucose levels 
48 
stimulates a rise in circulating insulin to induce translocation of glucose transporter 4 (GLUT4), resulting 
49 
in the uptake of glucose intake the cell.  Several other investigations (12-15) have also observed this 
50 
phenomenon of impaired insulin sensitivity upon acute exposure to HA in young healthy individuals. 
51 
Therefore, it is possible that insulin resistance at HA may be a mechanism suppressing exogenous 
52 
carbohydrate oxidation. However, little is known regarding alterations in intramuscular processes that 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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may regulate alterations in glucose uptake into the cell at HA compared to SL.  Investigation is required 
1 
on molecular pathways that regulate insulin dependent and independent translocation of GLUT4 to 
2 
better understand the potential mechanism that may govern alterations in carbohydrate metabolism.   
3 
 
4 
 
5 
 
6 
B3.  MILITARY RELEVANCE    
7 
 
8 
Service members deployed to HA regions of the world experience aerobic performance decrements. 
9 
Based on findings from our previous study, one contributing factor, at least during acute exposures to 
10 
HA, is a decreased ability to oxidize exogenous glucose, with no difference in total and endogenous 
11 
carbohydrate metabolism. During the initial exposure to HA there appears to be a diminished insulin 
12 
sensitivity in response to carbohydrate consumption, resulting in a 46% reduction in exogenous glucose 
13 
oxidation during exercise compared to SL.   
14 
Additionally, consumption of carbohydrate at HA did 
15 
not minimize declines in physical performance 
16 
compared to a matched non-nutritive placebo as 
17 
determined by a 2-mile time trial (Figure 3).  These 
18 
data indicate that while dietary carbohydrate or 
19 
carbohydrate supplementation may not support 
20 
exercise performance during acute HA exposure.   
21 
Though it was observed that exogenous glucose 
22 
oxidations was lower during exercise at HA compared 
23 
to SL, the mechanism leading to diminished exogenous 
24 
glucose oxidation at HA remains unclear.  The purpose 
25 
of this study is to define the metabolic underpinnings 
26 
for lower exogenous glucose oxidation to develop 
27 
appropriate dietary recommendations and/or 
28 
countermeasures to support acute HA military 
29 
operations.   
30 
 
31 
B4.  OBJECTIVES/SPECIFIC AIMS/RESEARCH QUESTIONS   
32 
 
33 
Objectives 
34 
 
35 
Confirm results from our previous investigation that acute HA exposure decreased exogenous 
36 
carbohydrate oxidation during steady-state aerobic exercise compared to SL, and explore if the 
37 
mechanism inhibiting plasma glucose uptake is insulin dependent or independent. 
38 
 
39 
Hypotheses 
40 
 
41 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Exogenous carbohydrate oxidation will be lower at HA compared to SL during steady-state aerobic 
1 
exercise at the same absolute workload, and potential regulation of diminished glucose uptake will be 
2 
examined by insulin dependent or independent mechanisms. 
3 
 
4 
B5.  RESEARCH PLAN    
5 
 
6 
       B5.1  Research Design   
7 
 
8 
This study will be a randomized crossover control trial. 
9 
 
10 
      B5.2  Research Subjects/Population(s) 
11 
 
12 
      B5.2.1  Subject Population(s)   
13 
 
14 
Subject population will be representative of active duty male service members, being in good health 
15 
and recreationally active.  Only males are being studied so that results from the current study can be 
16 
appropriately compared to our previous study (USARIEM protocol 16-02HC).  It is apparent, that sex-
17 
based differences exist during low-to-moderate intensity endurance exercise under at SL.  Several 
18 
investigations (16-19) have reported that females oxidize proportionately more fat and less 
19 
carbohydrate than males during aerobic exercise.  Sex-based differences in substrate oxidation during 
20 
exercise at SL may result in differences in response to change in substrate oxidation at HA between 
21 
men and women.  Based on findings from the current study, follow-up work may be conducted to 
22 
assess sex differences in change in substrate oxidation during exercise at HA. 
23 
 
24 
B5.2.2  Number of Subjects, Records, and/or Specimens 
25 
 
26 
We estimate that 30 individuals will require screening in order to complete testing on the 10 volunteers 
27 
necessary to reach statistical power.  All screening will stop once complete data has been collected on 
28 
10 volunteers.  Records and specimen collection are described in the Research Procedures and Data 
29 
Collection sections.  During briefings and the consenting process potential volunteers will be informed 
30 
that even though they may be eligible and want to participate, if we are able to obtain enough data from 
31 
preceding subjects, they may not ultimately be tested.      
32 
     
33 
     B5.2.3  Inclusion Criteria 
34 
 
35 
 
Men aged 18 – 39 years 
36 
 
Born at altitudes less than 2,100 m (~7,000 feet; Examples include Santa Fe, New Mexico; 
37 
Laramie, Wyoming; Etc.) 
38 
 
Physically active based on assessment of physical activity history (2-4 days per week aerobic 
39 
and/or resistance exercise) 
40 
 
Have supervisor approval (permanent party military and civilians) 
41 
 
Willing to refrain from alcohol,  smokeless nicotine products and dietary supplement use during 
42 
study periods 
43 
 
Refrain from taking any nonsteroidal anti-inflammatory drugs (NSAIDs; e.g. aspirin, Advil®, Aleve®, 
44 
Naprosyn®, or any aspirin-containing product) for 10 days before and at least 5 days AFTER each 
45 
muscle biopsy. (*Tylenol® or acetaminophen is ok to use if needed for discomfort) 
46 
 
47 
    B5.2.4  Exclusion Criteria 
48 
 
49 
 
Born at altitudes greater than 2,100 m (~7,000 feet; Examples include Santa Fe, New Mexico; 
50 
Laramie, Wyoming; Etc.) 
51 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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 
Living in areas that are more than 1,200 m (~4,000 feet), or have traveled to areas that are more 
1 
than 1,200 m for five days or more within the last 2 months (Examples include Ft. Huachuca, 
2 
Arizona; Lima, Peru; Feldberg, Germany, Etc.) 
3 
 
Musculoskeletal injuries that compromise exercise capability 
4 
 
Metabolic or cardiovascular abnormalities (determined by resting ECG), gastrointestinal disorders 
5 
(e.g., kidney disease, diabetes, cardiovascular disease, etc.)  
6 
 
Medication that affects macronutrient metabolism (i.e., diabetes medications, statins, 
7 
corticosteroids, etc) and/or the ability to participate in strenuous exercise  
8 
 
Evidence of apnea or other sleeping disorders 
9 
 
Prior diagnosis of high altitude pulmonary edema (HAPE) or high altitude cerebral edema (HACE) 
10 
 
Presence of asthma or respiratory tract infections (< 1 month prior) 
11 
 
Allergies or intolerance to foods (including but not limited to lactose intolerance/milk allergy), 
12 
vegetarian practices, or medications (including, but not limited to, lidocaine ) to be utilized in the 
13 
study 
14 
 
Smoking or vaping 
15 
 
History of complications with lidocaine  
16 
 
Taking medications that interfere with oxygen delivery and transport (Includes sedatives, sleeping 
17 
aids, tranquilizers and/or any medication that depresses ventilation, diuretics, alpha and beta 
18 
blockers) 
19 
 
Evidence of any physical, mental, and/or medical conditions that would make the proposed studies 
20 
relatively more hazardous as determined by OMSO   
21 
 
Present condition of alcoholism, anabolic steroids, or other substance abuse issues 
22 
 
Anemia (HCT <38% and HBG <12.5 g/dL) and Sickle Cell Anemia/Trait 
23 
 
Abnormal PT/PTT test or problems with blood clotting  
24 
 
Blood donation within 8 weeks of beginning the study 
25 
 
26 
      B5.3  Research Procedures   
27 
 
28 
Study Design 
29 
This randomized crossover study will examine substrate metabolic responses to ingesting 
30 
supplemental carbohydrate during steady-state aerobic-type exercise at sea level (SL) and following 
31 
acute (~5 h) exposure to HA (4,300 m) conditions in 10 healthy, recreationally active adults between 
32 
the ages of 18-39 yrs (Table 1).  Following a 48-hr muscle glycogen normalization period, volunteers 
33 
will complete 80-min of metabolically-matched, steady-state aerobic exercise on a treadmill, and 
34 
consume 145 g of glucose (1.8 g·min-1) at SL and HA.  Treadmill exercise will be performed at the 
35 
same absolute workload, with speed and grade being the same at SL and HA to induce the same 
36 
absolute workload between phases.  SL and HA trials will occur in the USARIEM hypobaric chamber 
37 
and will be separated by a minimum 7-d washout period between each protocol day.  6-6-[2H2] glucose 
38 
will be used as a tracer to assess glucose turnover.    Indirect calorimetry, breath sampling for 13C/12C 
39 
expired in CO2, and urine collections will be used to determine carbohydrate, fat, and protein oxidation 
40 
during exercise at SL and HA.  Serial blood draws will be collected during each trial to assess 
41 
endocrine and circulating substrate responses to exercise, carbohydrate, and hypoxia.  Muscle biopsies 
42 
will be collected before and after steady-state exercise to examine intramuscular glucose transport 
43 
expression and translocation, glycogen status, and activity enzyme intermediates in aerobic and 
44 
anaerobic energy metabolism.   
45 
 
46 
 
47 
 
48 
 
49 
 
50 
 
51 
 
52 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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1 
 
2 
 
3 
Table 1. Study Timeline 
4 
 
5 
Note that this is a potential timeline, specific study days and length of time to complete the study may be 
6 
dependent on volunteer’s availability, so total time commitment may be longer.  
7 
 
8 
Research Procedures 
9 
Anthropometric Data 
10 
Anthropometrics, performed using standardized techniques and equipment, will be used to determine 
11 
volunteer eligibility and characterize study volunteers.  Height will be measured to the nearest 0.1 cm 
12 
using a stadiometer at screening. Body mass will be measured after an overnight fast (≥ 8 hr), using a 
13 
calibrated digital scale to the nearest 0.1 kg at screening.  Body mass will be measured at baseline and 
14 
the morning of protocol days. 
15 
 
16 
Body composition will be determined using dual energy x-ray absorptiometry (DEXA, DPX-IQ, GE 
17 
Lunar Corporation, Madison, WI).  The DEXA technique allows for the non-invasive assessment of soft 
18 
tissue composition by region with a precision of 1-3% (20). The volunteer will lay face-up on the DEXA 
19 
densitometer table in shorts, t-shirts, and stocking feet.  Volunteers will be asked to remain motionless 
20 
for the 8-10 min scan. These data will be used to calculate total body mass, fat-free mass, and fat 
21 
mass. Calibration to external standards will be performed before actual data collection. The operator 
22 
remains in the room with the volunteer during the scan.  Measurements of body composition will be 
23 
used for participant characterization and to assess if fat mass and fat-free mass are confounding 
24 
variables for substrate oxidation at SL and HA.  
25 
 
26 
Determination of Peak Oxygen Uptake 
27 
Volunteers will complete two separate maximal aerobic exercise sessions to determine peak oxygen 
28 
uptake (V̇ O2peak) on a treadmill.  Two treadmill V̇ O2peak assessments will be conducted because HA 
29 
exposure of 4,300 m produces a decrease in V̇ O2peak of ~27% compared to SL (21).  As such, 
30 
volunteers will complete one treadmill V̇ O2peak at SL and one at HA so V̇ O2peak is known under both 
31 
conditions.  V̇ O2peak will be determined using an indirect, open circuit respirator system (True Max 2400, 
32 
Parvomedics, Sandy, Utah, USA).  Volunteers will be instructed to fast overnight (≥ 8 hr) before testing.  
33 
Volunteers will be clothed in appropriate athletic attire and perform this assessment at standard 
34 
ambient indoor temperature (20-22C) and humidity conditions (30-80%).  Volunteers will be given 
35 
adequate time to become familiar with the testing procedures and allowed a 3-min self-paced warm-up 
36 
on the treadmill. At the start of testing, the volunteer will put on a nose clip and a mouthpiece connected 
37 
to a 2-way respiratory valve, which is attached to a head piece to hold it in place. The volunteers will 
38 
Study Day 
SV 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
Medical Screening 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Body Composition 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Fitness Assessment 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood Sampling 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Exercise Practice Sessions 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
Muscle Biopsies 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Carbohydrate Depletion Exercise 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
 
Steady-State Exercise 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Study Diet 
 
 
 
 
 
X 
X 
 
 
 
 
 
X 
X 
 
 
Carbohydrate Tracer Study 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Post Biopsy Follow-Up 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Page 14 of 34 
 
begin by running for 4 min at a pace predetermined as comfortable at a 0% grade. At 4-min, the grade 
1 
will be increased to 4% followed by an additional 2% every 2 min thereafter until volitional exhaustion.  
2 
 
3 
Heart rate will be monitored using a heart-rate monitor (Polar Electro Inc, Oulu, Finland) the last 30 
4 
seconds of each workload during all testing.  The test will be stopped immediately if the subject reports 
5 
angina-like symptoms, exertional syncope, shows signs of poor perfusion (i.e., light-headedness, 
6 
confusion, ataxia, pallor, cyanosis, nausea, or cold and clammy skin), or if testing equipment fails.   
7 
 
8 
Steady-State Treadmill Exercise Prescription  
9 
Results from V̇ O2peak assessment will be used to prescribe steady-state treadmill exercise for protocol 
10 
days.  At SL and HA volunteers will complete 80-min of steady-state exercise on a treadmill at 55 ± 5% 
11 
of their VO2peak determined at HA, performing the same absolute work under both study conditions.  The 
12 
speed and grade of the treadmill will be determined using the ACSM metabolic equation for walking 
13 
based on desired V̇ O2 (22).  Prior to the protocol day, participants will perform a practice session of the 
14 
treadmill exercise to confirm the prescribed speed and grade are appropriate to induce target V̇ O2.  The 
15 
speed and grade determined during the practice session will be used at SL and HA to induce the same 
16 
absolute V̇ O2 between phases. 
17 
 
18 
Glycogen Normalization Period 
19 
A glycogen depletion protocol will be completed on a cycle ergometer, with intensity based on results 
20 
from VO2peak assessment.  After the warm-up period volunteers will complete 2-min of high-intensity 
21 
cycling (work period) at 90% VO2peak, followed by 2-min recovery period where volunteers will cycle at 
22 
50% VO2peak.  This work-to-recovery ratio will be maintained until volunteers are no longer able to 
23 
complete 2-min of cycling at 90% VO2peak, determined as the inability to maintain a cycling cadence of 
24 
60 rpm for 15-sec.  Cycling intensity will then be lowered to 80% VO2peak.  When the volunteer is unable 
25 
to complete 2-min of cycling at 80% VO2peak, cycling intensity will be lowered to 70% VO2peak.   Once the 
26 
volunteer can no longer complete 2-min of cycling at 70% VO2peak, cycling intensity will be lowered to 
27 
60% VO2peak.   The glycogen depletion protocol will be terminated once the volunteer is unable to 
28 
complete 2-min of cycling at 60% VO2peak.  For each drop in the intensity of the work period, the cycling 
29 
intensity during the recovery period will be maintained at 50% VO2peak for 2-min.  This exercise protocol 
30 
will maximally deplete muscle glycogen stores (23).  Prior to the protocol day, volunteers will perform a 
31 
practice session to ensure they are familiar with the glycogen depletion protocol procedures.  Following 
32 
familiarization volunteers will complete a glycogen depletion protocols 48-hrs before testing. 
33 
 
34 
After completing the glycogen depletion protocol, volunteers will be fed a controlled diet providing at 
35 
least 6.0 g carbohydrate ∙kg-1∙d-1 (~55-65% of total energy consumed) to ensure adequate glycogen 
36 
repletion and homogeneous glycogen levels within and across volunteers between phases (SL and 
37 
HA).  All food and beverages (except water) will be prepared and provided by study dietitians and 
38 
consist largely of military combat ration and supplemental food items.  Since 13C will be measured from 
39 
breath on protocol days, volunteers will be instructed to avoid foods high in natural abundance of 13C, 
40 
such as corn and cane sugar.  Diets provided during the two day normalization phase will not contain 
41 
these products.  
42 
 
43 
Altitude Chamber 
44 
To simulate ascension to high altitude volunteers will perform one arm of this crossover study in a 
45 
hypobaric (altitude) chamber under temperate (20°C, 30-50% rh; 12-16°C°C Wet Globe Bulb 
46 
Temperature (WGBT)) ambient conditions.  The chamber will reduce barometric pressure, and thus 
47 
ambient oxygen pressure, to a value similar to that at the summit of Pike Peak, CO (4,300 m).  
48 
Volunteers will be exposed to hypobaric/hypoxic conditions for 5-hrs before beginning steady-state 
49 
exercise to mimic our past investigations.  Total time in the chamber on the protocol day will be ~7-8-
50 
hrs.  All activities within the altitude chamber will be monitored by trained study staff.  The chamber is 
51 
on a regular maintenance schedule and chamber performance is documented each test day.  
52 
 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Exercise Protocol Day 
1 
Two catheters will be placed into the lower arm (one in each arm).  One arm will be used for infusion of 
2 
6,6-[2H2] glucose tracer and the other will be used for blood sampling under resting and exercise 
3 
conditions.  Following an initial blood and breath sample collection to determine background 
4 
enrichments, a primed, continuous infusion of 6,6-[2H2] glucose will begin (prime, 82.2 µmol∙kg-1; 
5 
continuous rate, 0.78 µmol∙kg-1∙min-1, Figure 4) (24, 25).  Volunteers will complete 80-min of steady-
6 
state exercise on a treadmill at 55 ± 5% of their VO2peak determined at HA, performing the same 
7 
absolute work under both study conditions.  V̇ O2, V̇ CO2, HR, and SaO2 will be measured at 
8 
approximately 320, 330, 340, 360, 380, and 395 min.  During exercise volunteers will consume a CHO 
9 
beverage at 1.8 g∙min-1 to examine the impact of altitude exposure on exogenous carbohydrate 
10 
oxidation efficiency.  Drinks will contain 145 g glucose, with a total volume of 1450 mL.  The drink will 
11 
be consumed in four boluses throughout the 80-min of steady-state exercise (320, 340, 360, and 380-
12 
min).  The first bolus will be 550 mL, with the remaining three being 300 mL.  The CHO beverage 
13 
consumed during exercise will be enriched with 200 mg U-13C-glucose (Cambridge Isotope Laboratory, 
14 
Andover, MA, USA) to increase the isotopic enrichment well above natural levels and optimize the 
15 
measurement of exogenous carbohydrate oxidation.   Before exercise, during the HA phase, volunteers 
16 
will be exposed to hypobaric hypoxic conditions for 5-hrs to mimic our previous investigation.  Muscle 
17 
samples will be obtained using percutaneous muscle biopsy, taken from the vastus lateralis using a 5-
18 
mm Bergstrom needle with manual suction while the volunteer is under local anesthesia (1% lidocaine) 
19 
according to the approved USARIEM SOP (26, 27). Muscle samples will be collected before (300 min) 
20 
and immediately (420 min) after 80-min of steady state exercise during both the SL and HA protocol 
21 
study days. The two muscle biopsy collections will occur from one incision each protocol day.  
22 
Following a minimum 7-d washout period, volunteers will return to the laboratory to complete the 
23 
second arm of the investigation.  A total of four muscle biopsies will be conducted per volunteer, two 
24 
per protocol day, with one incision made on each leg.          
25 
 
26 
 
27 
 
28 
Figure 4: Exercise Protocol Day 
29 
   
 
30 
       
31 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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Page 16 of 34 
 
 
1 
 
2 
 
3 
B5.4  Data Collection   
4 
 
5 
Blood Sampling 
6 
Blood samples will be collected using intravenous access by a USARIEM credentialed phlebotomist on 
7 
throughout protocol days under SL and HA conditions (Figure 4).  Baseline blood sampling will occur 
8 
after a 10-hr overnight fast.  A total of 26 blood draws will be completed during this study taking 438 mL 
9 
of blood sampled from each volunteer over the two protocol days (Table 2).  Blood samples will be 
10 
used for isotope analysis, assessment of nutrient status, hormone response, and circulating microRNA 
11 
(Table 3).   
12 
   
13 
Table 2. Total Volume of Blood Sampled for Protocol Days 
14 
Tube 
Number of Tubes 
Total Volume (mL) 
7.5 mL Red Top Tube 
8 
60 
4 mL Red Top Tube 
8 
32 
4 mL Green Top Tube 
16 
64 
1.2 mL Green Top Tube 
16 
20 
6 mL Purple Top Tube 
18 
108 
4 mL Purple Top Tube 
28 
112 
2.6 mL Gray Top Tube 
16 
42 
Total 
110 
438 
 
15 
 
16 
Table 3. Blood Analytes 
17 
 
 
 
 
Time Point (min) 
Analyte 
120 
180 
210 
240 
260 
280 
300 
320 
340 
360 
380 
400 
420 
Glucose1 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
Insulin2 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
Free fatty acids2 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
Lactate2 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
Glycerol2 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
Norepinephrine2 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
Epinephrine2 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
Hemoglobin1 
x 
 
 
 
 
 
x 
 
 
x 
 
 
x 
Hematocrit1 
x 
 
 
 
 
 
x 
 
 
x 
 
 
x 
13C-glucose3 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
 
6,6-[2H2] 
glucose4 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
c-miRNA*1 
x 
 
 
 
 
 
x 
 
 
x 
 
 
x 
Exosomal 
miRNA1 
x 
 
 
 
 
 
x 
 
 
x 
 
 
x 
PBMC+ 
x 
 
 
 
 
 
x 
 
 
 
 
 
x 
Archive 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
* Analytes: c-miRNA, circulating microRNA, +Peripheral blood mononuclear cell 
18 
   
19 
Samples will be analyzed at 1USARIEM, 2Pennington Biomedical Research Center (Baton Rouge, LA) 
20 
3Metabolic Solutions (Nashua, NH), and 4The Center for Translational Research in Aging and Longevity 
21 
(Little Rock, AR).  All blood samples will be separated into plasma and serum through centrifugation, 
22 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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aliquoted, and stored at –80˚C until analysis or shipment. Study samples will be collected and aliquoted 
1 
at USARIEM and specific samples will be shipped on dry ice to Pennington Biomedical Research 
2 
Center, Metabolic Solutions and the Center for Translational Research in Aging and Longevity.  Blood 
3 
sent out to other laboratories for analysis will not have any remaining samples after analysis is 
4 
completed.   USARIEM will retain all archive samples. 
5 
 
6 
Blood Sample Processing for Enrichment 
7 
Blood samples for 6,6-[2H2] glucose analysis will be collected at 180, 210, 240, 260, 280, 300, 320, 340, 
8 
360, 380, 400, and 420-min to calculate plasma glucose turnover at rest and in response to exercise 
9 
and carbohydrate intake under SL and HA conditions.  Serial blood sampling for analysis will be 
10 
collected in EDTA-coated tubes for plasma collection.  The tracer/tracee ratio (6,6-[2H2] 
11 
glucose/glucose) will be measured on the pentaacetate derivative by gas-chromatography-mass 
12 
spectrometry (GCMS; Agilent Technologies, Santa Clara, CA) (24). 
13 
 
14 
Blood samples for 13C-glucose analysis will be collected at 320, 340, 360, 380, and 400-min to 
15 
calculate the rate of plasma glucose oxidation.  13C/12C in plasma glucose will be measured using 
16 
isotope-ratio mass spectroscopy (Metabolic Solutions, Inc., Nashua, NH). 
17 
 
18 
Breath Sample Processing for Isotopic Enrichment 
19 
The natural enrichment of the glucose ingested and the 13C/12C in expired gas samples will be analyzed 
20 
using isotope-ratio mass spectroscopy (Metabolic Solutions, Inc., Nashua, NH). Breath samples will be 
21 
collected at approximately 320, 330, 340, 350, 360, 365, 370, 375, 380, 385, 390, and 395-min to 
22 
determine exogenous carbohydrate oxidation using single-patient breath collection bags (Quin-Tron 
23 
Instrument Company, Milwaukee, WI, USA). A baseline breath sample will also be collected prior to 
24 
correct for background of naturally occurring 13C. All breath samples will be transferred to 10 mL 
25 
evacuated tubes.    
26 
 
27 
Plasma Glucose Turnover Calculations 
28 
For calculation of plasma glucose turnover the Steele equation with modifications for non-steady state 
29 
will be used (28).  Enrichment (E) will be expressed as mole percent excess (MPE); calculated as 
30 
(TTR)/(1 + TTR), where TTR is the tracer to tracee ratio. Appropriate corrections for skewed 
31 
abundance distribution and overlapping spectra will be made for the TTR of the glucose tracers, 6,6-
32 
[2H2] glucose and U-13C-glucose (28).  From these calculations, total glucose Ra will be comprised of 
33 
rates of appearance of exogenous (i.e., ingested) glucose and of endogenous (i.e., hepatic glucose 
34 
production and negligible renal glucose production or splanchnic glucose) glucose (28): 
35 
 
36 
Total glucose Ra (Total Ra) = (F – ((pV x ((C2 + C1)) / 2) x ((E2 - E1) / (t2 - t1)))) / ((E2 + E1) / 2) 
37 
Glucose Rd = Total Ra – (pV (C2 - C1) / (t2 - t1)) 
38 
Exogenous glucose Ra (Exo Ra) = (( Total Ra + F ) x ( ( G2 + GE1) / 2 ) + ( (pV x ( (C2 + C1) / 2) ) x 
39 
((G2 - G1) / (t2 - t1)))Endogenous glucose Ra = Total Ra - Exo Ra 
40 
Metabolic Clearance Rate (MCR) = Glucose Rd / ((C2 + C1) / 2)   
41 
 
42 
Where F represents the infusion rate of 6,6-[2H2] glucose; pV is the effective volume of distribution for 
43 
glucose, C1 and C2 are plasma glucose concentrations at t1 and t2, respectively, E1 and E2 are plasma 
44 
enrichments of 6,6-[2H2] glucose at t1 and t2, respectively, and Ed and Ep are tracer enrichments of U-
45 
13C-glucose from the test drink and plasma, respectively.  
46 
 
47 
Assessment of Urinary Nitrogen Excretion 
48 
Volunteers will void urine before starting steady state exercise, with a subsequent urine sample 
49 
immediately after (400-min) the conclusion steady-state exercise to measure nitrogen excretion during 
50 
exercise.  Urine volume will be measured and aliquots stored at -20ºC until analyzed. Total nitrogen 
51 
content of the urine will be determined using pyrochemiluminescence (Antek 9000, Houston, TX).  
52 
 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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Calculations of Carbohydrate, Fat, and Protein Oxidation 
1 
Carbohydrate, fat, and protein oxidation rates will be calculated from V̇ O2 (L/min), V̇ CO2 (L/min), and 
2 
urinary nitrogen excretion rate (g/min) during the 80-min exercise bout as described by Jeukendrup and 
3 
Wallis (29): 
4 
 
5 
Fat oxidation (g/min) = 1.695 x V̇ O2 – 1.701 x V̇ CO2 – 1.77 x urinary nitrogen  
6 
Carbohydrate oxidation (g glucose/min) = 4.210 x V̇ CO2 – 2.962 x V̇ O2 – 2.37 x urinary nitrogen  
7 
Protein oxidation (g/min) = 6.25 x urine nitrogen excreted (g)/ urine collection time (min) 
8 
 
9 
Calculations of Exogenous and Endogenous Glucose Oxidation 
10 
Exogenous and plasma glucose oxidation will be calculated as (1): 
11 
 
12 
Exogenous glucose (g/min) = V̇ CO2 [(Rexp - Rref)/(Rexo - Rref)]/k 
13 
Plasma glucose (g/min) = V̇ CO2 [(Rexp - Rref)/(Rglu - Rref)]/k 
14 
 
15 
where V̇ CO2 is in L/min, Rexp is the observed isotopic composition of expired CO2, Rref is the isotopic 
16 
composition of expired CO2 at rest before ingestion of the first dose of 13C-glucose, Rexo is the isotopic 
17 
composition of the exogenous glucose ingested, Rglu is the isotopic composition of plasma glucose, 
18 
and k (0.747 L/g) is the volume of CO2 provided by the complete oxidation of glucose. Total 
19 
endogenous glucose oxidation can be calculated by subtracting exogenous glucose oxidation from total 
20 
CHO oxidation.  Endogenous glucose oxidation derived from muscle and liver can be determined by 
21 
subtracting plasma glucose oxidation from total carbohydrate oxidation (muscle), and subtracting 
22 
exogenous carbohydrate oxidation (liver) from plasma glucose oxidation (1).  The first 40 min of steady-
23 
state exercise will allow for equilibration between the 13C/12C in expired CO2 and the 13C/12C in CO2 
24 
produced in tissues (30). Thus, endogenous glucose oxidation will only be calculated from samples 
25 
obtained in the last 40 min of steady-state exercise (40 to 80 min). 
26 
 
27 
Muscle Glycogen 
28 
Approximately 20 mg of muscle will be dehydrated in a freeze dryer.  Samples will then be ground to 
29 
powder and visible connective tissue will be removed.  Powdered muscle will be placed in 500 µl 2 N 
30 
HCl.  Samples will then be placed in an incubator for 120 min at 100˚C.  Following incubation samples 
31 
will be neutralized with 1500 µl 0.67 N NaOH and glycogen will be quantified by a fluorometric assay 
32 
(Sigma-Aldrich, St. Louis, MO, USA).      
33 
 
34 
Immunofluorescence staining, microscopy, and image analysis 
35 
To determine GLUT4 translocation to the cellular membrane in response to exercise and carbohydrate 
36 
intake immediately after sample collection by muscle biopsy, excess blood and any visible collagen 
37 
and/or fat will be removed and muscle will be embedded in Tissue Tek Optimum Cutting Temperature 
38 
(OCT) compound (VWR International, Leicester, UK) and frozen in liquid nitrogen.  Frozen muscle 
39 
biopsy samples will be cryosectioned to a thickness of 5 μm onto uncoated glass microscope slides 
40 
(VWR International). Sections will be fixed in 75% acetone and 25% ethanol solution for 5 min, then 
41 
washed 3 times for 5 min in phosphate-buffered saline (PBS). A previously validated (31, 32) GLUT4 
42 
and dystrophin primary antibody (Abcam, Cambridge, UK) will be applied to muscle sections at a 1:200 
43 
and 1:400, respectively, dilution in 5% normal goat serum (Invitrogen, Carlsbad, CA, USA) for 2-hr at 
44 
room temperature.  Dystrophin staining will allow for visualization of the plasma membrane to 
45 
determine GLUT4 location.  Following primary antibody incubation, sections will be washed 3 times for 
46 
5 min in PBS and then incubated in 1:200 dilutions of secondary antibodies AlexaFluor 488-conjugated 
47 
goat anti-rabbit IgG (Invitrogen; GLUT4) and  AlexaFluor 594-conjugated goat anti-mouse IgG2b 
48 
(Invitrogen; Dystrophin) for 30 min at room temperature. Sections will then be washed 3 times for 5 min 
49 
in PBS and glass coverslips will be mounted with 20 μL mowiol mounting medium [6 g glycerol (Sigma-
50 
Aldrich), 2.4 g mowiol 4–88 (Sigma-Aldrich) and 0.026 g 1,4-Diazabicyclo[2.2.2]octane (DABCO) 
51 
(Sigma-Aldrich) dissolved in 18 mL 0.2 M Tris-buffer (pH 8.5) (Sigma-Aldrich)]. Images will be captured 
52 
using a Nikon TI-U inverted microscope equipped with a Retiga 2000R camera and NIS-Elements 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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image analysis software (Nikon, Tokyo, Japan) to determine colocalization of GLUT4 and dystrophin.  
1 
Quantitation of GLUT4 in the plasma membrane (dystrophin-stained region) will be performed using 
2 
Imaging software. 
3 
 
4 
mRNA and microRNA Expression 
5 
To understand potential molecular regulation of changes in substrate metabolism during exercise at SL 
6 
and HA this study will examine alterations in expression profiles of mRNA and microRNA that are 
7 
associated with substrate metabolism.  Total RNA will be isolated from approximately 25 mg of muscle 
8 
using a mirVanaTM miRNA isolation kit (Invitrogen, Carlsbad, CA, USA).  Quantity and quality of RNA 
9 
will be assessed using a Nanodrop ND-1000spectrophotometer (Nanodrop, Wilmington, DE, USA).  
10 
Equal amounts of total RNA will be synthesized into cDNA for analysis of mRNA (High-Capacity cDNA 
11 
RT Kit, Applied Biosystems, Foster City, CA, USA) and a TaqMan® microRNA RT kit (Applied 
12 
Biosystems).  Individual primers will be used to determine the mRNA expression of known intracellular 
13 
targets regulating substrate metabolism, through glycolysis, TCA cycle, fatty acid oxidation, and 
14 
glucose uptake.  Individual primers will be used to determine microRNA expression of microRNA that 
15 
have been reported to impact metabolism within the muscle.   
16 
 
17 
Circulating miRNA will be extracted from serum using previously published methodologies (33).  Due to 
18 
the small amount of RNA in the serum, 3.5 µL of a Spike-In Control (C. elegans miR-39; Qiagen) A pre-
19 
amplification step will be performed after reverse transcription to increase cDNA template using a 
20 
primer pool of 20 X Taqman® Small RNA Assays (Applied Biosystems) for miRNA of interest at 0.05X 
21 
concentration in 1X TE buffer.  All serum miRNA will be normalized to the geometric of external (Spike-
22 
In Control C. elegans miR-39) and internal controls to allow for both technical and inter-individual 
23 
normalization (34). Geometric mean of controls will be used to correct for possible outlying values and 
24 
abundance differences between the different controls (35).   
25 
 
26 
All reverse transcription for mRNA and miRNA, and pre-amplification of serum miRNA will be 
27 
conducted in a T100TM Thermal Cycler (Bio-Rad, Hercules, CA). A StepOnePlusTM real-time PCR 
28 
system (Applied Biosystems) will be used to perform all mRNA and miRNA analysis.  Fold changes will 
29 
be calculated using the ∆∆ cycle threshold (∆∆CT) method as described below in statistical analysis 
30 
section. 
31 
 
32 
Western Blotting 
33 
To understand potential molecular regulation of changes in substrate metabolism during exercise at SL 
34 
and HA this study will examine alterations in phosphorylation and total protein content of signaling 
35 
proteins associated with substrate metabolism.  Following previously published methods (36), ~30 mg 
36 
of muscle will be homogenized in ice-cold buffer to extract proteins for Western blotting.  Protein 
37 
concentration of supernatant (lysate) will be determined using 660 nm Protein Assay (ThermoFisher 
38 
Scientific, Waltham, MA, USA).  Phosphorylation status and total protein expression of molecular 
39 
markers associated with regulating substrate metabolism, through glycolysis, TCA cycle, fatty acid 
40 
oxidation, and glucose uptake.  Muscle lysates will be solubilized in Laemmli buffer, with equal amounts 
41 
of total protein (15 μg) separated by SDS-PAGE using precast Tris·HCl gels (Bio-Rad). Proteins will be 
42 
transferred to polyvinylidene fluoride (PVDF) membranes and exposed to commercially available 
43 
primary antibodies at 4°C overnight.  Labeling will be performed using secondary antibody (anti-rabbit 
44 
IgG conjugate with horseradish peroxidase; Cell Signaling Technology), and chemiluminescent reagent 
45 
will be applied (Super Signal, West Pico Kit; Pierce Biotechnology, Rockford, IL, USA). Blots will be 
46 
quantified using a phosphoimager (ChemiDoc XRS; Bio-Rad) and Image Lab software (Bio-Rad). To 
47 
confirm equal protein loading per well a normalizing protein such as GAPDH or HSP90 will be 
48 
assessed.  
49 
 
50 
Enzyme Activity 
51 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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Colorimetric enzyme activity assays will be conducted to assess the impact of altitude exposure on 
1 
substrate energy metabolism, to include but not limited to pyruvate dehydrogenase (PDH) and citrate 
2 
synthase.   
3 
 
4 
Pyruvate dehydrogenase (PDH) activity will be measured from approximately 10 mg of muscle 
5 
homogenized in 100 μl of ice-cold PDH Assay Buffer (Sigma-Aldrich).  Samples will be loaded into a 96 
6 
well clear plate with PDH developer (Sigma-Aldrich) and incubated at 37°C for 3 min.  Initial PDH 
7 
activity will be read at 450 nm.  Samples will continue to be incubated at 37°C and read every 5 min 
8 
until the highest standard value (12.5 nmol) is met.  A final absorbance measurement will be taken at 
9 
450 nm. Change in absorbance will be calculated from initial and final read. 
10 
 
11 
Homogenate from muscle samples prepared for Western blotting will be used to assess citrate 
12 
synthase activity, by combining 10 μl of diluted (1:10; 0.1 M Tris HCl pH 8.1) sample to 150 μl of 
13 
reaction master mix (1 mL DNTB, 3 mg Acetyl CoA, and 8 mL 0.1 M Tris HCl pH 8.1). The reaction will 
14 
be initiated when 10 μl of 10 mM oxaloacetate will be added to each well (37). Samples will be read at 
15 
412 nm. Data will be normalized to protein content of muscle homogenate. 
16 
 
17 
Enzyme activity will be determined using a colorimetric assay analyzed on an SpectraMax® M Series 
18 
Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). 
19 
 
20 
Any use of the data or samples outside of this defined research plan will be submitted as a protocol 
21 
amendment or a new protocol. 
22 
 
23 
      B5.5  Managing Data and/or Human Biological Specimens for this Research   
24 
 
25 
All data and medical information obtained will be considered privileged and held in confidence.  Study 
26 
volunteers will be assigned unique subject identification (ID) numbers that will not contain any personal 
27 
identifiers such as name, social security number, address, date of birth, zip code, etc.  This study 
28 
subject ID number will be used on all data collection instruments, to include questionnaires, data 
29 
collection forms, computer records, etc.  A number will be assigned as each volunteer is medically 
30 
cleared for participation.  A master list linking the volunteers’ names and ID numbers will be kept in a 
31 
separate locked file in the principal investigator's office, or kept in a computer file with password-
32 
protected access restricted to the principal investigator.  When the results of the research are published 
33 
or discussed in conferences, no information will be included that would reveal identity.  Study samples 
34 
will be processed on site at USARIEM.  All samples will be stored using the subject identification 
35 
number.  De-identified blood and urine will be shipped on dry ice to PBRC for analysis.  De-identified 
36 
samples for isotopic analysis will be shipped on dry ice to UAMS (blood) and Metabolic Solutions (blood 
37 
and breath).  All samples will be shipped via FedEx and stores in these laboratories until analyzed.  
38 
Once analyzed, there will be no remaining sample for storage.  
39 
 
40 
Only personnel assigned to the research study by the principal investigator will have access to the data.  
41 
Hard copy data records will be stored for a minimum of three years from the time the study is 
42 
completed.  Electronic data records will be maintained for a period of at least five years after the study 
43 
has been completed.   
44 
 
45 
      B5.6  Managing Data and/or Human Biological Specimens for Future Research 
46 
 
47 
 The master list linking subjects with their data will be destroyed when the study is closed. De-identified 
48 
study samples will be stored in -80ºC freezer at USARIEM in room 322 or 304 for potential future use.  
49 
All samples will be de-identified and maintained indefinitely. Only personnel assigned to the research 
50 
study by the principal investigator will have access to samples. The de-identified data and samples will 
51 
remain under the control of the PI and may be shared with outside collaborators for future research.   
52 
Any use of the samples outside of this defined protocol will be submitted as a protocol amendment or a 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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new protocol.   
1 
 
2 
      B5.7  Devices, Drugs, Dietary Supplements, Nutritional Supplements, And Biologics   
3 
 
4 
      B5.7.1  Devices 
5 
 
6 
5.7.1.1 FDA-approved device being used in this research according to the approved 
7 
labeling. DEXA, DPX-IQ, Lunar Corporation, True Max 2400, Parvomedics, Sandy, Utah, USA, 
8 
Polar Electro Inc, Oulu, Finland 
9 
 
10 
5.7.1.2 FDA-approved device being used in this research in a manner other than its 
11 
approved labeling 
12 
N/A 
13 
 
14 
B5.7.2  Drugs 
15 
N/A 
16 
 
17 
B5.7.2.1 FDA-approved and used in accordance with the approved labeling  
18 
N/A 
19 
  
20 
B5.7.2.2 FDA-approved and used in a manner not in accordance with its approved labeling 
21 
N/A 
22 
 
23 
B5.7.2.3 Any drug not approved by the FDA 
24 
N/A 
25 
 
26 
     B5.8  Statistical Analysis 
27 
 
28 
               B5.8.1  Sample Size Estimation 
29 
 
30 
Statistical power and sample size were determined from the effect size of exogenous 
31 
carbohydrate oxidation (-14 ± 8 g/40-min exercise) that we previously observed at HA compared 
32 
to SL. Using this information the sample size necessary to determine statistical significance 
33 
between intervention arms is 7, with an alpha of 0.05 and 80% power.  To account for greater 
34 
variance in the response to altitude between volunteers and increased study power to 90%, 10 
35 
volunteers will be enrolled to complete this investigation.  Based on our previous studies we 
36 
request to consent 30 individuals to complete 10.  
37 
 
38 
               B5.8.2  Data analysis   
39 
 
40 
Statistical analyses will be conducted using either SPSS (IBM Corp. Armonk, NY), SAS 9.3 (SAS 
41 
Institute Inc., Carey, NC), or equivalent.  Common descriptive statistics will be used to describe 
42 
volunteer characteristics.  Shapiro-Wilk tests will be used to determine normality of data.  Paired t-
43 
test will be performed to determine main effects of phase (SL versus HA) for glucose turnover and 
44 
glucose oxidation.  Mixed model repeated measures ANOVA will be performed to determine main 
45 
effects of time (baseline versus post-exercise), phase (SL versus HA) and time-by-phase 
46 
interactions for glucose and insulin concentrations, GLUT4 translocation, glycogen, mRNA and 
47 
microRNA, Western blots and enzyme activity.   If interactions are significant, appropriate post-
48 
hoc correction will be used to examine these relationships.  Correlation coefficients and multiple 
49 
regression analysis will be used to evaluate relationships between study outcome measures.  The 
50 
alpha level will be adjusted for multiple comparisons, with the level for statistical significance set 
51 
at P < 0.05.   
52 
 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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1 
 
2 
 
3 
SECTION C:  HUMAN RESEARCH PROTECTIONS 
4 
 
5 
C1.  RECRUITMENT AND CONSENT   
6 
 
7 
       C1.1  Identification and Selection of Subjects   
8 
 
9 
Potential Volunteers will be recruited from the U.S. Army Natick Soldier Systems Center (NSSC) pool of 
10 
Human Research Volunteers (HRVs).  Additional volunteers will be recruited from civilian population 
11 
and active duty military stationed at NSSC, as well as civilians from the local surrounding area using 
12 
flyers and electronic posting, such as newsletters and social media.  In addition, other military 
13 
organizations may be recruited through coordination with NSSC Soldier and Squad Optimization & 
14 
Integration Team (SSOI-T).  The SSOI-T will schedule the consent briefing for the military research 
15 
volunteers outside the NSSC and Ms. Katelyn Guerriere will serve as the ombudsman for non-HRV 
16 
Soldiers.    
17 
 
18 
       C1.2  Recruitment Process   
19 
 
 
20 
Superiors of Service members (e.g., unit officers, senior NCOs, and equivalent civilians) / supervisors 
21 
of DoD civilians (e.g., military and civilian supervisors or anyone in the supervisory structure) will not be 
22 
present at any recruitment sessions or during the consent process in which members of units under 
23 
their command / personnel under their supervision are afforded the opportunity to participate as human 
24 
subjects of research.   
25 
 
26 
For Soldiers in the NSSC pool of military volunteers, the Principal Investigator will furnish a copy of the 
27 
consent form to the Human Research Volunteer Program Coordinator or designee. The Coordinator will 
28 
schedule the consent briefing for the military human research volunteer platoon and will serve as 
29 
ombudsman during the briefing.  Civilians and permanent party military will be recruited for the study 
30 
using flyers posted or electronically distributed or through informational briefs.  Principal Investigator or 
31 
Project Coordinator will receive and respond to inquiries submitted from these recruitment materials, 
32 
and will schedule informed consent briefings for these potential volunteers. Katelyn Guerriere will be 
33 
the ombudsman for group briefings of non-HRV Soldiers, the HRV coordinator will be the ombudsman 
34 
for HRV briefings and all civilian briefings will be done one-on-one. 
35 
 
36 
       C1.3  Eligibility   
37 
 
38 
Volunteers will be medically cleared by the Office of Medical Support and Oversight (OMSO) before 
39 
participation in accordance with USARIEM procedures outlined for screening volunteers for research 
40 
involving exercise, muscle biopsies, and altitude exposure either at USARIEM or at the potential 
41 
volunteer’s duty station. Potential military and civilian volunteers will undergo the same clearances used 
42 
at USARIEM for research involving maximal aerobic exercise testing and high altitude exposure. In 
43 
addition, volunteers will be screened for problems with blood clotting, including prothrombin time (PT)/ 
44 
partial thromboplastin time (PTT), which is a specific criterion for research involving muscle biopsies. 
45 
Health problems identified during the screening process will be documented and a copy provided to the 
46 
volunteer. The volunteer will be encouraged to make an appointment with their primary care provider 
47 
for a full evaluation of the problem. Volunteers with evidence of any physical, mental, and/or medical 
48 
conditions that would make the proposed studies relatively more hazardous will be excluded. Any 
49 
personal health information collected during this screening process will be destroyed at the time of 
50 
study withdrawal or at the completion of the study. 
51 
 
52 
All volunteers must be willing to consume only food and beverages provided by study staff during the 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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48-hr normalization period and on protocol days.  Additionally, volunteers must be willing to refrain from 
1 
any additional exercise during this period.   
2 
 
3 
       C1.4  Consent Process   
4 
 
5 
Informed consent documents will be provided to each prospective volunteer in writing, as well as in an 
6 
oral presentation by the principal investigator or his designee.  The purpose of this study, procedures 
7 
involved, risks, and expectations of volunteers will be explained.  The principal investigator or designee 
8 
will answer all group and private questions.  Interested volunteers will sign the informed consent form 
9 
prior to undergoing initial screening for which they will have blood drawn for study-specific clearance.  If 
10 
they meet all the medical selection criteria after completing the screening health assessment they will be 
11 
scheduled to begin data collection.  A copy of the informed consent will be provided to the volunteer with 
12 
the original kept for study documentation.  No study procedures will occur prior to the volunteer giving 
13 
informed consent.  Volunteers who have already consented will be informed of any new information or 
14 
changes to the protocol that may affect their willingness and ability to continue participation in the study 
15 
using an approved consent addendum.    
16 
 
17 
C1.4.1  Research involving subjects with cognitive impairment or who lack capacity to 
18 
provide informed consent: N/A 
19 
 
20 
                 C1.4.2  Research involving non-English speaking subjects: N/A 
21 
 
22 
C1.4.3  Research involving a waiver of the requirement to obtain informed consent OR 
23 
alteration of the elements of informed consent: N/A 
24 
 
25 
C1.4.4  Research involving a waiver of the requirement for investigator to obtain a signed 
26 
consent form: N/A 
27 
 
28 
                 C1.4.5  Waivers of assent or parental permission when the research involves children: 
29 
N/A 
30 
 
31 
                 C1.4.6  Research involving data collection for the USAMRMC Volunteer Registry  
32 
Database 
33 
 
34 
It is the policy of USAMRMC that data sheets are to be completed on all volunteers participating in 
35 
greater than minimal risk research for entry into the U.S. Army Medical Research and Materiel 
36 
Command Volunteer Registry Database.  The information to be entered into this confidential database 
37 
includes name, address, social security number, study name, and dates.  The intent of the database is 
38 
twofold: first, to readily answer questions concerning an individual’s participation in research sponsored 
39 
by the USAMRMC; and second, to ensure that the USAMRMC can exercise its obligation to ensure 
40 
research volunteers are adequately warned (duty to warn) of risks and to provide new information as it 
41 
becomes available.  The information will be stored at the USAMRMC for a minimum of 75 years. 
42 
 
43 
C2.  COMPENSATION FOR PARTICIPATION   
44 
 
45 
Military and civilian personnel will receive $25 for each successful blood draw.  There are 26 blood draws 
46 
during the two protocol days (13 per protocol day).  Volunteers completing all 26 draws will receive $650.  If 
47 
a volunteer does not complete the entire study, they will be compensated for the number of successful 
48 
blood draws they did complete.  Volunteers will not be eligible for any other form of compensation during 
49 
this study.    
50 
 
51 
Military personnel will have paperwork submitted to petition for hazardous duty pay for performing any 
52 
testing under hypobaric hypoxic conditions.  
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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1 
C3.  WITHDRAWAL FROM RESEARCH PARTICIPATION   
2 
 
3 
Volunteers will be allowed to withdraw at any time without penalty or loss of benefits to which they would 
4 
otherwise be entitled.  If a participant wishes to withdraw mid-study while the chamber is mimicking hypoxic 
5 
conditions they will enter a separate portion of the chamber that will allow them to descend back to SL 
6 
conditions.  Once descent is completed the participant will be allowed to leave the chamber. An 
7 
investigator may stop an individual’s participation in the study if the participant is unwilling or unable to 
8 
complete study procedures.  An investigator or the medical monitor may also withdraw a participant if the 
9 
individual becomes ill or injured or it would not be in the participant’s best interest to continue.   
10 
 
11 
C4.  PRIVACY FOR SUBJECTS   
12 
 
13 
To protect the participant’s privacy, any of their research-related records will be labeled or “coded” with an 
14 
assigned research participant number that will not include their name or social security number. Dr. Lee M. 
15 
Margolis will keep the link between participant number and the participant’s research records in a locked 
16 
cabinet.  Any documents that will require the participant’s name, such as the consent form, will be kept in a 
17 
locked cabinet separate from any research documents that contain the participant’s ID number.  The 
18 
principal investigator is the only person who will be able to match the research participant number with any 
19 
of their personal identifying information.    
20 
 
21 
When the results of the research are published or discussed in conferences, no information will be included 
22 
that would reveal the volunteers identity to others.  If photographs, videos, or audio-tape recordings of you 
23 
will be used for educational purposes, your identity will be protected or disguised.  All identifiable or 
24 
recognizable information (e.g., names and faces) will be covered in any photographs unless volunteers 
25 
agree to sign a photo release form. 
26 
 
27 
C5.  CONFIDENTIALITY PROCEDURES FOR RESEARCH RECORDS, DATA, HUMAN BIOLOGICAL 
28 
SPECIMENS   
29 
 
30 
All data and medical information obtained will be considered privileged and held in confidence.  Study 
31 
volunteers will be assigned unique subject identification (ID) numbers that will not contain any personal 
32 
identifiers such as name, social security number, address, date of birth, zip code, etc.  This study subject 
33 
ID number will be used on all data collection instruments, to include questionnaires, data collection forms, 
34 
computer records, etc.  A number will be assigned as each volunteer is medically cleared for participation.  
35 
A master list linking the volunteers’ names and ID numbers will be kept in a separate locked file in the 
36 
principal investigator's office, or kept in a computer file with password-protected access restricted to the 
37 
principal investigator and project manager.  The master list linking subjects with their data will be destroyed 
38 
when the study is closed. When the results of the research are published or discussed in conferences, no 
39 
information will be included that would reveal identity.  Study samples will be processed on site at 
40 
USARIEM.  All samples will be stored using the subject identification number.  Samples will be shipped for 
41 
analysis to Pennington Biomedical Research Center, Metabolic Solutions, and The Center for Translational 
42 
Research in Aging and Longevity.  Remaining samples will be stored at USARIEM in a -80ºC freezer in 
43 
room 322 or 304.  The volunteers name or other identifiable information will not be included on any data, 
44 
data collection sheets, specimens, or other research records.  De-identified samples will be maintained 
45 
indefinitely. 
46 
 
47 
Only personnel assigned to the research study by the principal investigator will have access to the data.  
48 
Hard copy data records will be stored for a minimum of three years from the time the study is completed.  
49 
Electronic data records will be maintained for a period of at least five years after the study has been 
50 
completed.   
51 
 
52 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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Date: 11 February 2019 
 
 
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C6.  RISKS OF HARM, MEASURES TO REDUCE THE RISKS OF HARM, AND BENEFITS OF 
1 
PARTICIPATION 
2 
 
3 
       C6.1 Risks of Harm   
4 
Research Procedure Name:  DEXA Scan 
5 
Research Procedure Description:  Volunteer will lay face-up on the DEXA densitometer table in shorts, 
6 
t-shirts, and stocking feet.  Volunteers will be asked to remain motionless for the 8-10 min scan. 
7 
Research-related Risks:  The DEXA scan is an X-ray and is considered to be a low risk procedure.  
8 
The radiation dose of the whole-body DEXA scan is 0.1 mrem.  This dose is equivalent to 
9 
approximately 1/250 of normal annual background radiation, 1/9 of the radiation received in a 
10 
transatlantic flight, or 1/30 of the radiation received in a chest X-ray.     
11 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  A quality assurance check will be 
12 
completed on the DEXA each day prior to its use; the software will not allow the use of the DEXA 
13 
densitometer if the quality assurance check fails.   
14 
 
15 
Research Procedure Name:  Indwelling Catheters 
16 
Research Procedure Description:  A needle will be used to guide a catheter into the antecubital vein of 
17 
the volunteer.  The catheter will be attached to saline to keep the line patent for multiple blood draws.     
18 
Research-related Risks:  The risks of blood sampling are small and usually limited to local bruising or 
19 
swelling.  Also sometimes volunteers feel faint or may faint.  If the volunteer has had problems with 
20 
fainting during blood draws in the past, they may be more prone to them during future procedures.  If 
21 
the catheter, the tube that is left in the arm after the needle is removed, becomes clogged at any time 
22 
during the protocol, we will have to replace this to continue blood sampling and therefore the study. 
23 
This will require another needle to be inserted into your arm.  In addition, the catheter can cause 
24 
irritation, bruising, swelling, infection, or an allergic reaction. 
25 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Trained technicians will use sterile 
26 
techniques to place the catheter; however, in spite of being careful there is a chance that the site may 
27 
become infected.  Volunteers should not donate blood for eight weeks before or after this study.   
28 
 
29 
Research Procedure Name:  Venipuncture 
30 
Research Procedure Description: A needle will be used for single blood draws of the antecubital vein.   
31 
Research-related Risks:  Venipuncture is a routine clinical procedure the medical community 
32 
commonly uses to obtain blood samples.  The immediate complications may be slight pain during the 
33 
entry of the needle into the skin, possible dizziness, and syncope.   Dizziness or syncope constitutes 
34 
no long-term harm, and immediate relief is achieved by having the subject put their head down 
35 
between their knees or lie down.  Additionally, a hematoma may result from the venipuncture, but this 
36 
is more unsightly than risk producing.  Late complications might include thrombosis of the vein due to 
37 
trauma or infection.  These complications are extremely rare. 
38 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Participant monitoring, aseptic 
39 
technique, including sterile disposable blood collection apparatus and adherence to standard medical 
40 
precautions reduce risk.  Trained technicians will perform all venipuncture. 
41 
 
42 
Research Procedure Name:  Lidocaine Injection 
43 
Research Procedure Description:  Roughly 8-10 mL of Lidocaine will be injected using a needle at the 
44 
site of the incision, superficially (i.e., skin) and within the vastus lateralis. 
45 
Research-related Risks:  Slight pain at the site of injection might occur. Although rare, anaphylactic 
46 
reactions may also occur following administration of lidocaine.  Unlikely, but possible side effects could 
47 
include: dizziness, confusion, shakiness, visual changes, nausea, and unusually slow heartbeat.   
48 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Volunteers will be instructed to 
49 
Notify study coordinator or PI immediately if an allergic (i.e., swelling, itching, rash, hives, difficulty 
50 
swallowing, or difficulty breathing) reaction occurs. In the case of severe reaction, lidocaine use will be 
51 
discontinued.                 
52 
 
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        Research Procedure Name:  Muscle Biopsy 
1 
Research Procedure Description: A small incision will be made in the skin and fascia of the vastus 
2 
lateralis.  A 5-mm Bergstrom biopsy needle will pass through these incisions with manual suction while 
3 
the volunteer is under local anesthesia (1% lidocaine) to collect muscle samples. 
4 
Research-related Risks:  Percutaneous needle muscle biopsies have been established as a non-
5 
routine, but safe research procedure. Similar to blood draws, there is a risk that volunteers will feel 
6 
faint or may faint.  If the participant has had problems with fainting during blood draws or muscle 
7 
biopsies in the past, they may be more prone to them during future procedures.  There is some risk of 
8 
post-biopsy infection, which can be minimized by employing correct sterile procedures and carefully 
9 
instructing volunteers on care of the wound.  Moderate stiffness, hematoma and swelling around the 
10 
biopsy site may occur following the procedure, but this usually resolves itself within several days.  
11 
Some minimal scarring will accompany healing of the incision and formation of a hypertrophic scar or 
12 
keloid is possible.  Although this is a rare event in fair-skinned persons, the incidence of hypertrophic 
13 
scarring or keloid formation associated with healing of a primarily closed skin biopsy site (i.e., one 
14 
which was closed with sutures immediately afterward) is 5-10% in dark-skinned persons.  
15 
Complications of bleeding can be reduced by applying direct pressure to the wound following the 
16 
biopsy.  If symptoms should occur, they usually do not interfere with normal walking or heavier 
17 
exercise.  Volunteers with evidence of bleeding diatheses or with local skin infection or irritation, or 
18 
recent anticoagulant medication (including aspirin), will not be used.     
19 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Volunteers will be instructed about 
20 
precautions against hematoma and infection.  They will be given a handout outlining instructions for 
21 
proper care of the incision site (Appendix A).  Dr. Margolis or Dr. Pasiakos will perform the procedure 
22 
with sterile technique according the USARIEM Muscle Biopsy SOP and record notes for each biopsy 
23 
(Appendix B).  The medical staff at USARIEM will follow-up with volunteers within 3 days post-biopsy 
24 
to monitor for any sign of infection, bleeding, or hematoma.  To minimize the likelihood of hypertrophic 
25 
scarring or keloid formation, biopsy wounds will be closed as promptly as feasible. 
26 
 
27 
Research Procedure Name:  Altitude Exposure 
28 
        Research Procedure Description: Participants will exercise at 4300 m simulated altitude.    
29 
 Research-related Risks:  Hypoxemia, lightheadedness, AMS, HAPE, HACE, ear pain/discomfort, and 
30 
peripheral/facial edema. 
31 
Measures to Minimize Risks of Harm: (Precautions, safeguards):   
32 
 
During hypobaric exposures, volunteers will always be accompanied by one or more trained 
33 
staff that will monitor signs and symptoms of research-related risks. To additionally minimize 
34 
risk, volunteers will be familiarized with safety procedures and equipment in the hypobaric 
35 
chamber. 
36 
 
Hypoxemia- Exposure to hypobaric hypoxia introduces the hazard of hypoxemia.  Depending 
37 
upon the degree and duration of hypobaric exposure, hypoxemia may be well tolerated without 
38 
consequences, or may present a full spectrum of clinical entities ranging from mild discomfort 
39 
to potentially lethal conditions. Participants will be monitored by study staff and participants will 
40 
be allowed to terminate testing early if they desire. 
41 
 
Lightheadedness- Exercising at altitude can cause an increased risk of lightheadedness which 
42 
may occur just after stopping heavy exercise. Lightheadedness increases the risk that the 
43 
volunteer could fall. This risk (rare) will be minimized by having at least one staff member in 
44 
close proximity to the volunteer (as is done for all exercise tests regardless of location) so that 
45 
he/she will not fall off.  If a volunteer becomes lightheaded and feels faint, he/she will be 
46 
assisted off the treadmill and instructed to lay down with feet up until fully recovered. 
47 
Volunteers will be assisted on and off exercise and other experimental apparatus as needed.  
48 
 
AMS- AMS is a self-limited syndrome that is common when unacclimatized lowlanders are 
49 
exposed to higher altitudes. Symptoms of AMS include headache, nausea, anorexia, lethargy, 
50 
dizziness, tiredness, weakness, insomnia, and sometimes vomiting. The prevalence and 
51 
severity of AMS increase directly in proportion to ascent rate and elevation. With rapid ascent, 
52 
and no beneficial treatment (e.g., breathing supplemental O2) symptoms commonly appear 
53 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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within four to six hours. Symptoms of AMS will reach their severity within 18 to 24 hrs of 
1 
altitude exposure, and be the most severe after awakening. With rapid ascent to the altitudes of 
2 
3000 m and 4000 m for a 24-hr period, more than 30-90% of the untreated volunteers will likely 
3 
experience AMS, with symptoms ranging from mild (10 to 40%), to moderate (20 to 40%) to 
4 
severe (10 to 20%). Due to the duration (~7-8 hrs) of the altitude exposure in the current study, 
5 
AMS may occur but should not reach its most severe symptoms.  
6 
 
HAPE/ HACE- The incidence of clinical HAPE in unacclimatized sea-level residents being 
7 
exposed to high altitude for at least a few days appears to be less than 1%. As a clinical 
8 
condition, the possibility of HACE occurring when sea-level residents are exposed to high 
9 
altitude appears to be even lower than the incidence of HAPE. If either or both are recognized 
10 
early and treated by descent, they are completely reversible. With regard to the proposed 
11 
study, the risk of any volunteer developing either HAPE or HACE is considered remote and is 
12 
not expected. If a volunteer shows developing symptoms of HAPE or HACE they will be 
13 
removed from the simulated high altitude environment.  
14 
 
Ear Pain/Discomfort- Changes in ambient gas pressure during induction of or return from 
15 
hypobaric conditions can cause untoward effects due to gas trapped in the body.  At the 
16 
relatively slow rates of pressure change during normal operation (simulated ascent and/or 
17 
descent) of the hypobaric chamber, some individuals may experience discomfort in their ears, 
18 
paranasal sinuses, teeth, or abdomen.  In healthy individuals, pressure in the middle ear and 
19 
sinuses can be equalized with ambient atmospheric pressure by swallowing, yawning, or 
20 
tensing the muscles of the throat, procedures that contract the pharyngeal muscles and open 
21 
the Eustachian tubes, thereby ventilating the middle ear and sinus.  Another more effective 
22 
means of ventilating the middle ear and sinuses is forced expiration against a closed nose and 
23 
mouth.  Adjunctive therapy may also include the use of short-acting antihistamines or 
24 
decongestants to reduce swollen Eustachian tubes and sinus openings due to inflammation or 
25 
infection.  During recompression (descent), moderately severe ear pain may be experienced by 
26 
volunteers who cannot maintain Eustachian tube patency.  If ear or sinus pain occurs, 
27 
recompression will be stopped and the affected volunteer will be decompressed until the pain 
28 
resolves; recompression will then be restarted at a slower rate.  Damage to eardrums is likely 
29 
only under the unusual situation where deliberately rapid recompression is necessitated by dire 
30 
emergency.  
31 
 
Peripheral/Facial Edema- Peripheral and facial edema occurs in some individuals when initially 
32 
exposed to high altitude. It is characterized by pronounced edema of the face and upper 
33 
extremities, decreased urine output and weight gain. Although uncomfortable, it is a benign 
34 
condition. The peripheral and facial edema resolves with descent. 
35 
 
36 
Research Procedure Name:  Exercise 
37 
Research Procedure Description:  Exercise testing will occur on a cycle ergometer or treadmill.  
38 
Exercise will be at various levels of intensity based on exercise protocol. 
39 
Research-related Risks:  Exercise is generally considered safe and beneficial for individuals without 
40 
cardiovascular disease. The U.S. prevalence of fatal events is approximately 1:100,000 to 1:300,000 
41 
in competitive high school athletes and increases to 1:15,000 to 1:50,000 in athletes over the age of 
42 
35.  Current civilian and military guidelines state that individuals less than 40 years of age who have 
43 
no symptoms of or known presence of heart disease or major coronary risk factors have a low risk for 
44 
cardiac complications during vigorous exercise.  All volunteers in this study fall into this low risk 
45 
category.  Local muscle discomfort and fatigue may occur in active muscles during and shortly after 
46 
exercise.  Muscle soreness, ranging in intensity from mild to severe, may persist for 1 to 7 days.  
47 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Studies have confirmed the safety of 
48 
maximal exercise testing, particularly among apparently healthy persons without significant 
49 
cardiovascular risk factors.  As a precaution, there will be at least one spotter during all exercise 
50 
sessions, and heart rate will be monitored in real time during testing.  In addition, exercise monitors 
51 
and test administrators will be CPR-certified.   
52 
 
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Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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Research Procedure Name: 6,6-[2H2] glucose infusions  
1 
Research Procedure Description: 6,6-[2H2] glucose will be infused using a indwelling catheter and 13C-
2 
glucose will be consumed in a carbohydrate beverage. 
3 
Research-related Risks: The primary risks associated with tracer studies are those related to venous 
4 
catheterization. The catheter can cause irritation, bruising, or infection.  There are no known risks or 
5 
reported side effects associated with administration of 6,6-[2H2] glucose infusions to humans during 
6 
clinical or experimental studies.  The risks associated with the infusion include volume overload, 
7 
infection, and allergic reaction to the infused substance.  There have been no occurrences of volume 
8 
overload, no occurrences of infection or allergic reaction attributable to iodine used prior to 
9 
venipuncture in any of the 200 infusion protocols that the investigators have been involved.  To 
10 
minimize the likelihood of these events occurring, infusion rate will be closely monitored and 
11 
maintained at less than 30 ml/hr throughout the entire infusion protocol day. 
12 
Measures to Minimize Risks of Harm: All staff who directly participate in the infusion studies will be 
13 
properly trained how to safely monitor (i.e., infusion pumps and IV lines) infusion studies from Drs. 
14 
Margolis and Pasiakos, who have extensive experience with infusion studies.  In addition, infusates 
15 
will be prepared sterile, pyrogen-free, and in the proper dosages by a licensed pharmacist.  
16 
 
17 
Research Procedure Name: 13C-glucose ingestion 
18 
Research Procedure Description: 13C-glucose will be consumed in a carbohydrate beverage. 
19 
Research-related Risks: There are no known risks of consuming stable isotope of 13C-glucose. 13C is 
20 
naturally occurring in the diet (~1%). It is a nonradioactive isotope. A trace amount will be used to 
21 
establish carbohydrate oxidation (200 mg in 145 g of glucose, < 0.1%), which is a safe increase in 13C. 
22 
Measures to Minimize Risks of Harm: Pyrogen-free isotope will be purchased from reputable supplier. 
23 
       
24 
C6.2  Incidental or Unexpected Findings   
25 
 
26 
Health problems identified during the screening process will be documented and a copy provided to 
27 
the volunteer. The volunteer will be encouraged to make an appointment with their primary care 
28 
provider for a full evaluation of the problem. Volunteers with evidence of any physical, mental, and/or 
29 
medical conditions that would make the proposed studies relatively more hazardous will be excluded. 
30 
 
31 
      C6.3  Potential Benefits   
32 
 
33 
There is no health or other benefits related to participation in this study.  Information obtained from this 
34 
research may benefit other individuals in the future. 
35 
 
36 
C7.  DATA AND SAFETY MONITORING   
37 
 
38 
       C7.1 Monitoring 
39 
 
40 
The PI will, with the assistance of Associate Investigators, continuously evaluate recruitment, the 
41 
informed consent process, adverse events, and protocol adherence and deviations in order to identify 
42 
unanticipated problems or risks to the volunteers associated with the research. The PI will ensure that 
43 
the number of volunteers recruited for this study complies with the protocol.  Every morning, volunteers 
44 
will be asked the following questions to evaluate their readiness to test. 
45 
 
46 
• 
How have you been feeling well since the last test in our laboratory (below average, average, above 
47 
average)? 
48 
• 
Do you have any pain or symptoms to report that may affect our testing today (e.g., sinus 
49 
congestion, fatigue, muscle soreness, fever, tooth pain)? 
50 
• 
Have you reported all food and beverages consumed in the last 24 h that were not provided to you 
51 
by study staff? 
52 
• 
What time did you fall asleep last night and awake this morning? 
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• 
Did you perform any exercise or physical activities outside of study activities in the last 24 h? 
1 
 
2 
The PI or project manager will monitor data monthly during the data collection portion of the protocol.  A 
3 
log will be created listing any missing data.   
4 
 
5 
       C7.2 Research Monitor (as applicable)   
6 
The research monitor for this study is MAJ  Robin Cushing.  This individual is an appropriate subject 
7 
matter expert not associated with the protocol. The research monitor shall, at a minimum, review all 
8 
unanticipated problems involving risk to subjects or others, serious adverse events and all subject 
9 
deaths associated with the protocol and provide an unbiased written report of the event.  The PI and 
10 
research monitor will discuss “discontinuation criteria” for individual volunteers as the study progresses, 
11 
based on their observations of the volunteer during testing or non-testing periods.  Other 
12 
responsibilities may be assigned by the MRMC IRB as needed. 
13 
 
14 
C8.  REPORTABLE EVENTS    
15 
 
16 
       C8.1  Expected adverse events 
17 
 
18 
An adverse event is defined as any untoward or unfavorable medical occurrence in a human research 
19 
participant, including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, 
20 
or disease, temporally associated with the individual’s participation in the research, whether or not 
21 
considered related to the individual’s participation in the research. 
22 
 
23 
Expected adverse events are some level of AMS under HA conditions, potential fainting during blood 
24 
draws and muscle biopsies, local bruising and soreness from blood draws and muscle biopsies, and 
25 
muscle soreness or pain from exercise. Moderate stiffness, hematoma and swelling may occur around 
26 
the muscle biopsy site.  
27 
 
 
28 
All medical events that the USARIEM Office of Medical Support and Oversight (OMSO) evaluates will 
29 
be reported to the ORQC.  The PI will report all adverse events to the Research Monitor, if one was 
30 
appointed for the study.   
31 
 
32 
Expected adverse events which are not serious are reported to the IRB at the time of continuing review 
33 
of the protocol.   
34 
 
35 
       C8.2  Unexpected adverse events and unanticipated problems 
36 
 
37 
A serious adverse event is any adverse event temporally associated with the subject’s participation in 
38 
research that is fatal, life-threatening, permanently disabling, requires inpatient hospitalization, or 
39 
results in congenital anomalies/birth defect, overdose or cancer, or based on appropriate medical 
40 
judgment, may jeopardize the participant, or may require medical or surgical intervention to prevent one 
41 
of the above outcomes. 
42 
 
43 
All medical events will be reported to USARIEM’s Office of Medical Support and Oversight (OMSO). 
44 
OMSO staff will retain a copy of the report in the subject’s OMSO medical file as a means of tracking 
45 
and analyzing trends in medical events.  The PI will report all adverse events to the Research Monitor, 
46 
if one was appointed for the study. 
47 
 
48 
All unanticipated problems involving risk to subjects or others, serious adverse events that are 
49 
unexpected and determined to be at least possibly or definitely related to study participation, will be 
50 
promptly reported within one working day by phone (508-233-6306/4811) or email 
51 
(usarmy.natick.medcom-usariem.mbx.usariem-rqc-protocol@mail.mil) to the USARIEM ORQC  and the 
52 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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Commander.  These events will also be reported to the HQ USAMRMC IRB within one working day by 
1 
phone (301-619-6240), or by e-mail (usarmy.detrick.medcom-usamrmc.other.irb-office@mail.mil)  
2 
 
3 
Adverse events assessed by the PI as not serious and serious adverse events that are deemed to be 
4 
unrelated to participation in the study will be reported to the IRB at the time of continuing review of the 
5 
protocol.   
6 
 
7 
The research monitor is required to review all unanticipated problems involving risk to volunteers or 
8 
others, serious adverse events and all volunteer deaths associated with the protocol and provide an 
9 
unbiased written report of the event.  At a minimum, the research monitor should comment on the 
10 
outcomes of the event or problem, and in the case of a serious adverse event or death, comment on 
11 
the relationship to participation in the study.  The research monitor should also indicate whether he or 
12 
she concurs with the details of the report provided by the study investigator.  Reports for events 
13 
determined by either the investigator or research monitor to be: possibly related, unexpected, and 
14 
serious or suggest that the research places subjects or others at increased risk of harm during 
15 
participation, will be promptly forwarded to the ORQC and HQ USAMRMC IRB. 
16 
 
17 
In the event of a medical emergency at facilities on the Natick Soldier Center, the local Emergency 
18 
Medical Services (EMS) will be contacted immediately by dialing 5911. The installation security 
19 
personnel will direct the ambulance to the proper location on the installation.  While awaiting their 
20 
arrival, Basic Life Support will be rendered by study personnel or on-site medical coverage. EMS 
21 
response time to USARIEM is approximately 5 minutes.   Transport time to definitive care is 
22 
approximately 8 minutes. 
23 
      C8.3  Adverse device effects: N/A 
24 
  
25 
      C8.4  FDA-regulated research under IND and IDE: N/A 
26 
 
27 
 
28 
 
29 
SECTION D:  REFERENCES 
30 
 
31 
 
32 
1. 
O'Hara JP, Woods DR, Mellor A, Boos C, Gallagher L, Tsakirides C, Arjomandkhah NC, Holdsworth DA, 
33 
Cooke CB, Morrison DJ, et al. A comparison of substrate oxidation during prolonged exercise in men at 
34 
terrestrial altitude and normobaric normoxia following the coingestion of 13C glucose and 13C fructose. 
35 
Physiol Rep 2017;5(1). doi: 10.14814/phy2.13101. 
36 
2. 
Peronnet F, Massicotte D, Folch N, Melin B, Koulmann N, Jimenez C, Bourdon L, Launay JC, Savourey G. 
37 
Substrate utilization during prolonged exercise with ingestion of (13)C-glucose in acute hypobaric hypoxia 
38 
(4,300 m). Eur J Appl Physiol 2006;97(5):527-34. doi: 10.1007/s00421-006-0164-2 [doi]. 
39 
3. 
Jeukendrup A, Brouns F, Wagenmakers AJ, Saris WH. Carbohydrate-electrolyte feedings improve 1 h time 
40 
trial cycling performance. Int J Sports Med 1997;18(2):125-9. doi: 10.1055/s-2007-972607. 
41 
4. 
Jeukendrup AE. Carbohydrate intake during exercise and performance. Nutrition 2004;20(7-8):669-77. doi: 
42 
10.1016/j.nut.2004.04.017 [doi];S0899900704001169 [pii]. 
43 
5. 
Jeukendrup AE. Carbohydrate feeding during exercise. Eur J Sport Sci 2008;8(2):77-86. doi: 
44 
10.1080/17461390801918971. 
45 
6. 
Trommelen J, Fuchs CJ, Beelen M, Lenaerts K, Jeukendrup AE, Cermak NM, van Loon LJ. Fructose and 
46 
Sucrose Intake Increase Exogenous Carbohydrate Oxidation during Exercise. Nutrients 2017;9(2). doi: 
47 
10.3390/nu9020167. 
48 
7. 
Young AJ, Cymerman A, Burse RL. The influence of cardiorespiratory fitness on the decrement in maximal 
49 
aerobic power at high altitude. Eur J Appl Physiol 1985;54:12-5. 
50 
8. 
Young AJ, Reeves JT. Human adaptation to high terrestrial altitude. Edtion ed. In: Burr RE, Pandolf KB, eds. 
51 
Textbook of Military Medicine, Part III: Disease and the Environment, Volume: Medical Aspects of 
52 
Deployment to Harsh Environments. Washington, D.C.: Office of the Surgeon General, Department of the 
53 
Army, and Borden Institute, 1998:644-88. 
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Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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9. 
Roberts AC, Reeves JT, Butterfield GE, Mazzeo RS, Sutton JR, Wolfel EE, Brooks GA. Altitude and beta-
1 
blockade augment glucose utilization during submaximal exercise. J Appl Physiol (1985) 1996;80(2):605-15. 
2 
10. 
Brooks GA, Butterfield GE, Wolfe RR, Groves BM, Mazzeo RS, Sutton JR, Wolfel EE, Reeves JT. Increased 
3 
dependence on blood glucose after acclimatization to 4300 m. J Appl Physiol 1991;70:919-27. 
4 
11. 
Pirnay F, Crielaard JM, Pallikarakis N, Lacroix M, Mosora F, Krzentowski G, Luyckx AS, Lefebvre PJ. Fate of 
5 
exogenous glucose during exercise of different intensities in humans. J Appl Physiol Respir Environ Exerc 
6 
Physiol 1982;53(6):1620-4. 
7 
12. 
Larsen JJ, Hansen JM, Olsen NV, Galbo H, Dela F. The effect of altitude hypoxia on glucose homeostasis in 
8 
men. J Physiol-London 1997;504(1):241-9. doi: DOI 10.1111/j.1469-7793.1997.241bf.x. 
9 
13. 
Braun B, Rock PB, Zamudio S, Wolfel GE, Mazzeo RS, Muza SR, Fulco CS, Moore LG, Butterfield GE. 
10 
Women at altitude: short-term exposure to hypoxia and/or alpha(1)-adrenergic blockade reduces insulin 
11 
sensitivity. Journal of Applied Physiology 2001;91(2):623-31. 
12 
14. 
Oltmanns KM, Gehring H, Rudolf S, Schultes B, Rook S, Schweiger U, Born J, Fehm HL, Peters A. Hypoxia 
13 
causes glucose intolerance in humans. Am J Resp Crit Care 2004;169(11):1231-7. doi: 
14 
10.1164/rccm.200308-1200OC. 
15 
15. 
Peltonen GL, Scalzo RL, Schweder MM, Larson DG, Luckasen GJ, Irwin D, Hamilton KL, Schroeder T, Bell 
16 
C. Sympathetic inhibition attenuates hypoxia induced insulin resistance in healthy adult humans. J Physiol-
17 
London 2012;590(11):2801-9. doi: 10.1113/jphysiol.2011.227090. 
18 
16. 
Carter SL, Rennie C, Tarnopolsky MA. Substrate utilization during endurance exercise in men and women 
19 
after endurance training. Am J Physiol Endocrinol Metab 2001;280(6):E898-907. 
20 
17. 
Devries MC, Hamadeh MJ, Phillips SM, Tarnopolsky MA. Menstrual cycle phase and sex influence muscle 
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glycogen utilization and glucose turnover during moderate-intensity endurance exercise. Am J Physiol Regul 
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Integr Comp Physiol 2006;291(4):R1120-8. doi: 10.1152/ajpregu.00700.2005. 
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18. 
Horton TJ, Pagliassotti MJ, Hobbs K, Hill JO. Fuel metabolism in men and women during and after long-
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duration exercise. J Appl Physiol (1985) 1998;85(5):1823-32. 
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19. 
Phillips SM, Atkinson SA, Tarnopolsky MA, MacDougall JD. Gender differences in leucine kinetics and 
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nitrogen balance in endurance athletes. J Appl Physiol (1985) 1993;75(5):2134-41. 
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20. 
Lohman TG, Harris M, Teixeira PJ, Weiss L. Assessing body composition and changes in body composition. 
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Another look at dual-energy X-ray absorptiometry. Ann N Y Acad Sci 2000;904:45-54. 
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21. 
Young AJ, Cymerman A, Burse RL. The influence of cardiorespiratory fitness on the decrement in maximal 
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aerobic power at high altitude. Eur J Appl Physiol Occup Physiol 1985;54(1):12-5. 
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22. 
ACMS' Guidelines for Exercise Testing and Prescription: Seventh Edition. 2006. 
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23. 
McInerney P, Lessard SJ, Burke LM, Coffey VG, Lo Giudice SL, Southgate RJ, Hawley JA. Failure to 
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repeatedly supercompensate muscle glycogen stores in highly trained men. Med Sci Sports Exerc 
34 
2005;37(3):404-11. 
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24. 
Kim IY, Schutzler SE, Azhar G, Wolfe RR, Ferrando AA, Coker RH. Short term elevation in dietary protein 
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intake does not worsen insulin resistance or lipids in older adults with metabolic syndrome: a randomized-
37 
controlled trial. BMC Nutr 2017;3. doi: 10.1186/s40795-017-0152-4. 
38 
25. 
Kim IY, Williams RH, Schutzler SE, Lasley CJ, Bodenner DL, Wolfe RR, Coker RH. Acute lysine 
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supplementation does not improve hepatic or peripheral insulin sensitivity in older, overweight individuals. 
40 
Nutr Metab (Lond) 2014;11(1):49. doi: 10.1186/1743-7075-11-49. 
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26. 
Evans WJ, Phinney SD, Young VR. Suction applied to a muscle biopsy maximizes sample size. Med Sci 
42 
Sports Exerc 1982;14(1):101-2. 
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27. 
Pasiakos SM, McClung HL, McClung JP, Urso ML, Pikosky MA, Cloutier GJ, Fielding RA, Young AJ. 
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Molecular responses to moderate endurance exercise in skeletal muscle. Int J Sport Nutr Exerc Metab 
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2010;20(4):282-90. 
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28. 
Wolfe RR, Chinkes DL. Isotope tracers in metabolic research: principles and practice of kinetic analysis: John 
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Wiley & Sons, 2005. 
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Jeukendrup AE, Wallis GA. Measurement of substrate oxidation during exercise by means of gas exchange 
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measurements. Int J Sports Med 2005;26 Suppl 1:S28-37. doi: 10.1055/s-2004-830512. 
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Pallikarakis N, Sphiris N, Lefebvre P. Influence of the bicarbonate pool and on the occurrence of 13CO2 in 
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exhaled air. Eur J Appl Physiol Occup Physiol 1991;63(3-4):179-83. 
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Bradley H, Shaw CS, Bendtsen C, Worthington PL, Wilson OJ, Strauss JA, Wallis GA, Turner AM, 
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Wagenmakers AJ. Visualization and quantitation of GLUT4 translocation in human skeletal muscle following 
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glucose ingestion and exercise. Physiol Rep 2015;3(5). doi: 10.14814/phy2.12375. 
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Bradley H, Shaw CS, Worthington PL, Shepherd SO, Cocks M, Wagenmakers AJ. Quantitative 
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immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of 
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endurance and sprint interval training. Physiol Rep 2014;2(7). doi: 10.14814/phy2.12085. 
58 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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33. 
Margolis LML, S.J.; Ezzyat, Y.; Fielding, R.A.; Rivas, D.A. Circulating microRNA are predictive of aging and 
1 
acute adaptive response to resistance exercise in men. J Gerontol A Biol Sci Med Sci 2016;EPub Ahead of 
2 
Print. 
3 
34. 
Marabita F, de Candia P, Torri A, Tegner J, Abrignani S, Rossi RL. Normalization of circulating microRNA 
4 
expression data obtained by quantitative real-time RT-PCR. Brief Bioinform 2016;17(2):204-12. doi: 
5 
10.1093/bib/bbv056. 
6 
35. 
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate 
7 
normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. 
8 
Genome Biol 2002;3(7):RESEARCH0034. 
9 
36. 
Margolis LM, Rivas DA, Berrone M, Ezzyat Y, Young AJ, McClung JP, Fielding RA, Pasiakos SM. Prolonged 
10 
Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake 
11 
and Associated microRNA Expression. Front Physiol 2016;7:445. doi: 10.3389/fphys.2016.00445. 
12 
37. 
Lessard SJ, Rivas DA, Alves-Wagner AB, Hirshman MF, Gallagher IJ, Constantin-Teodosiu D, Atkins R, 
13 
Greenhaff PL, Qi NR, Gustafsson T, et al. Resistance to aerobic exercise training causes metabolic 
14 
dysfunction and reveals novel exercise-regulated signaling networks. Diabetes 2013;62(8):2717-27. doi: 
15 
10.2337/db13-0062. 
16 
 
17 
 
18 
 
19 
SECTION E:  ABBREVIATIONS AND ACRONYMS 
20 
HA; high altitude, SL; sea level, ATP; adenosine triphosphate, GLUT4; glucose transporter 4, PDHC; 
21 
pyruvate dehydrogenase complex, HIF-1α; hypoxic-inducible factor 1α, PDK; pyruvate dehydrogenase 
22 
kinase, TCA; tricarboxylic acid cycle, MORE; Modular Operational Ration Enhancement, CHO; 
23 
carbohydrate, DEXA; dual energy x-ray absorptiometry, HR; heart rate, SaO2; oxygen saturation, miRNA; 
24 
microRNA, TTR; tracer to tracee ratio, MPE; mole percent excess, PBR; phosphate-buffered saline, OCT; 
25 
Optimum Cutting Temperature, PVDF; polyvinylidene fluoride 
26 
 
27 
 
28 
 
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SECTION F: DoD PRIVACY RULE AND PROTECTED HEALTH INFORMATION (HIPAA) 
30 
 
31 
 
32 
 NA – institution is not a covered entity 
33 
 
34 
 NA – will not use or disclose protected health information 
35 
 
36 
 HIPAA authorization will be obtained  
37 
 
38 
 An application for waiver/alteration of HIPAA authorization will be submitted   
39 
 
40 
 
41 
 
42 
 
43 
 
44 
 
45 
 
46 
 
47 
 
48 
 
49 
 
50 
 
51 
 
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Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
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Date: 11 February 2019 
 
 
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2 
 
3 
 
4 
Appendices 
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Appendix A: Muscle Biopsy Care 
6 
 
7 
The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-
8 
state aerobic exercise   
9 
 
10 
PRINCIPAL INVESTIGATOR: 
11 
Lee M. Margolis, Ph.D. 
12 
 
13 
Muscle Biopsy Care 
14 
Information for the Participant 
15 
 
16 
You have just had a percutaneous muscle biopsy. The muscle biopsy site is closed with a steri-strip or 
17 
butterfly bandaid. This is covered with 4 x 4 gauze pad and then an elastic bandage. 
18 
 
19 
As the anesthetic wears off, you may feel a dull ache at the biopsy site for 1-3 days. Many people have no 
20 
pain at all. Instructions for the care of the biopsy site are as follows: 
21 
 
22 
1. You may perform all your normal activities immediately after the biopsy. 
23 
2. Leave the ACE bandage in place for 2 hours after the biopsy. After this time, remove everything 
24 
except the steri-strip or butterfly bandaid. 
25 
3. Do not get the incision wet for 24 hours after the biopsy.  
26 
4. Do not take any aspirin or aspirin-containing products for 5 days after the biopsy unless MD 
27 
approves. Also avoid ‘aspirin-like’ drugs such as Motrin. Indocin, etc. 
28 
S. After 2 days, no special precautions are required and the site should be almost completely 
29 
healed. 
30 
6. If any of the signs below occur, contact:  
31 
– Fever 
32 
– Bleeding from biopsy site 
33 
– Inflammation at biopsy site (warmth, redness, tenderness, pus formation, clear discharge, 
34 
opening of incision, lump under incision, or rash around incision). If you are not sure if it 
35 
is healing normally, alert study staff 
36 
– Persistent numbness in the leg 
37 
– Pain at the biopsy site more than one week after the biopsy 
38 
 
39 
 
40 
Lee M. Margolis, PhD, RD 
41 
U.S. Army Research Institute of Environmental Medicine 
42 
Building 42, Room 203 
43 
10 General Greene Ave 
44 
Natick, MA 01760 
45 
Office Phone: 508-233-4591 
46 
Cell Phone: 203-522-0624 
47 

Protocol Title:  The mechanistic effects of acute hypobaric hypoxia on exogenous carbohydrate utilization during steady-state aerobic exercise   
Version #:  3 
Date: 11 February 2019 
 
 
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5 
Appendix B: Muscle Biopsy Notes 
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7 
Participant ID: ______________ 
8 
 
9 
Trial    1   /   2  
10 
 
11 
Date: 
Biopsy One 
Leg: L    or    R 
Time: 
Lidocaine 
Size: 
Notes: 
 
Biopsy Two 
Leg: L    or    R 
Time: 
Lidocaine 
Size: 
Notes: 
 
 
12