Effects of Severe Negative Energy Balance on Inflammation During a Simulated Military Operation
Phase: NA
Conditions: Inflammation
Sponsor: United States Army Research Institute of Environmental Medicine

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
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PROTOCOL TITLE:  Effects of severe negative energy balance on inflammation, iron 
1 
absorption, nutritional status, skeletal muscle and whole-body metabolic homeostasis, cognitive, 
2 
and physical performance during a simulated sustained operation (SUSOPS) 
3 
 
4 
SECTION A:  RESEARCH TEAM AND LOCATIONS 
5 
 
6 
A1.  RESEARCH TEAM 
7 
 
8 
Study Role 
Institution/Company and Contact Information 
Sponsor 
Organization/Institution/Company:   
Military Nutrition Division (MND),  
US Army Research Institute of Environmental Medicine (USARIEM) 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Point of Contact: N/A   
 
Principal Investigator 
Name and Degree: Stefan M Pasiakos, PhD 
Title:  Nutritional Physiologist 
Institution:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-6474 
Email:  stefan.m.pasiakos.civ@mail.mil 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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Associate Investigator(s) 
 
Name and Degree:  James P McClung, PhD 
Title:  Nutrition Biologist 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4979 
Email:  james.p.mcclung8.civ@mail.mil 
Name and Degree:  Stephen R Hennigar, PhD 
Title:  Nutrition Biologist, ORISE Post-doctoral fellow 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4305 
Email: stephen.r.hennigar.ctr@mail.mil 
Name and Degree:  MAJ Nicholas D Barringer, PhD, RD 
Title:  Research Dietitian 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-6982 
Email: nicholas.d.barringer.mil@mail.mil 
Name and Degree:  Lee M Margolis, PhD, RD 
Title: ORISE Post-doctoral fellow  
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4291 
Email:  lee.m.margolis.ctr@mail.mil 
Name and Degree:  J Philip Karl, PhD, RD 
Title:  Research Physiologist 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5978 
Email:  james.p.karl.civ@mail.mil 
Name, Rank, and Degree: Harris R Lieberman, PhD  
Title:  Psychologist 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number: 508-233-4856 
Email: harris.r.lieberman.civ@mail.mil 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 3 of 59 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name, Rank, and Degree: Tracey J Smith, PhD, RD 
Title: Research Dietitian 
Institution/Company:  MND, USARIEM  
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number: 508-233-4868 
Email: tracey.smith10.civ@mail.mil 
Name, Rank, and Degree: Jennifer Rood, PhD 
Title: Associate Executive Director for Cores/Resources, Professor 
Institution/Company: Pennington Biomedical Research Center  
Address: 6400 Perkins Road, Baton Rouge, LA 70808 
Phone Number: 255-763-2524 
Email: Jennifer.rood@pbrc.edu 
Name and Degree:  Svein Martini, MS 
Title:  Principal Scientist 
Institution/Company:  Norwegian Defence Research  
Establishment (FFI) 
Address:  P.O. Box 25, NO-2027 Kjeller, Norway 
Phone Number:  (+47) 63 80 75 17 
Email: svein.martini@ffi.no 
Name and Degree:  Olav Vikmoen, PhD 
Title:  Post-doctoral Researcher 
Institution/Company:  Norwegian Defence Research  
Establishment (FFI) 
Address:  P.O. Box 25, NO-2027 Kjeller, Norway 
Phone Number:  (+47) 63 80 78 25 
Email: olav.vikmoen@ffi.no 
Name, Rank, and Degree:  Hilde Teien, MS 
Title:  Senior Scientist 
Institution/Company:  Norwegian Defence Research  
Establishment (FFI) 
Address:  P.O. Box 25, NO-2027 Kjeller, Norway 
Phone Number:  (+47) 63 80 76 52 
Email:  Hilde-Kristin.Teien@ffi.no 
Name, Rank, and Degree:  Jessica A. Gwin, PhD 
Title:  ORISE Post-doctoral fellow 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-6982 
Email:  Jessica.a.gwin.ctr@mail.mil 
Name, Rank, and Degree:  Alyssa N. Varanoske, PhD 
Title:  ORISE Post-doctoral fellow 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  (978) 328-6350 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 4 of 59 
 
 
 
 
Consultants 
 
Email:  alyssa.n.varanoske.ctr@mail.mil 
Name, Rank, and Degree: Rasha Hammamieh, PhD 
Title: Director, Integrative and Systems Biology 
Institution/Company: USACEHR 
Phone Number: 301-619-2338 
Email: rasha.hammamieh1.civ@mail.mil 
Name, Rank, and Degree: Graham S Finlayson, PhD  
Title:  Associate Professor 
Institution/Company:  University of Leeds 
Address: Institute of Psychological Sciences, University of Leeds,  
Leeds, LS2 9JT, UK 
Phone Number:  +44 (0) 113 343 5742 
Email: g.s.finlayson@leeds.ac.uk 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name, Rank, and Degree: Oshin Vartanian, PhD 
Title: Senior Scientist  
Institution/Company:  Defence Research and Development Canada 
Address: University of Canada 
Email: oshin.vartanian@drdc-rddc.gc.ca 
Name, Rank, and Degree:  John Caldwell, PhD 
Title: ORISE Post-doctoral fellow  
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Email: drjohncaldwell@gmail.com 
Other Key Research                         
Personnel (as applicable) 
 
Name, Rank, and Degree:  Adrienne Hatch, MS, RD 
Title:  Project Coordinator  
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-5648 
Email:  adrienne.m.hatch.civ@mail.mil 
 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 5 of 59 
 
Other Individuals Supporting  
the Research  
(as applicable) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Name, Rank, and Degree:  Nancy Murphy, MS 
Title:  Laboratory Manager 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4598 
Email:  nancy.e.murphy5.civ@mail.mil 
Name, Rank, and Degree:  Christopher Carrigan, BS 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5101 
Email: christopher.t.carrigan.civ@mail.mil 
 
Name, Rank, and Degree:  Patrick Radcliffe, BS 
Title: Research Assistant, ORISE Fellow 
Institution/Company: MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5958 
Email:  Patrick.n.radcliffe.ctr@mail.mil 
Name, Rank, and Degree:  Emily Howard, BS 
Title: Research Assistant, ORISE Fellow 
Institution/Company: MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  860-617-7620 
Email: emily.e.howard@uconn.edu  
Name, Rank, and Degree:  Marques Wilson, MS  
Title:  Research Physiologist 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5681 
Email:  marques.a.wilson.civ@mail.mil 
 
Name, Rank, and Degree:  Claire Whitney, MS, RD 
Title:  Research Dietitian 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4875 
Email:  claire.c.whitney.civ@mail.mil 
 
Name, Rank, and Degree: Heather Fagnant, MS, MPH, RD 
Title:  Research Dietitian, ORISE Fellow 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5958 
Email: heather.s.fagnant.ctr@mail.mil 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 6 of 59 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
Name, Rank, and Degree:  Nicholes Armstrong, MS, RD 
Title:  Research Dietitian 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-6492 
Email:  nicholes.j.armstrong.civ@mail.mil 
Name, Rank, and Degree: Susan McGraw, BS 
Title:  Research Nutritionist 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-6492 
Email:  susan.m.mcgraw6.civ@mail.mil 
Name, Rank, and Degree:  Anthony Karis, BS 
Title:  Research Physical Scientist 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4754 
Email:  anthony.j.karis.civ@mail.mil 
 
Name, Rank, and Degree:  SGT Cassandra Rousayne, MS 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address: 10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-5140 
Email:  cassandra.l.rousayne.mil@mail.mil 
Name, Rank, and Degree:  SPC Katakyie Sarpong, BS 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4835 
Email:  katakyie.p.sarpong.mil@mail.mil 
Name, Rank, and Degree:  Lauren Thompson, BS 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg. 42, Natick, MA 01760 
Phone Number:  508-233-4896 
Email:  lauren.a.thompson.civ@mail.mil 
Name, Rank, and Degree:  Philip Niro, BA 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Natick, MA 01760 
Phone Number:  508-233-5628 
Email:  philip.j.niro.civ@mail.mil 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 7 of 59 
 
 
                 Name, Rank, and Degree:  SPC Marcus Sanchez 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  240-893-3560 
Email:  marcus.a.sanchez20.mil@mail.mil 
 
Name, Rank, and Degree: SSG Stephen Mason 
Title: Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  540-718-4446 
Email:  Stephen.a.mason14.mil@mail.mil 
 
Name, Rank, and Degree:  SPC Cornal Pounds, BS 
Title:  Research Assistant 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  478-550-4235 
Email:  cornal.a.pounds.mil@mail.mil 
 
Name, Rank, and Degree: SPC Lauren Dare, BA 
Title: Research Assistant 
Institution/Company: MND, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 803-261-3389 
Email: lauren.e.dare2.mil@mail.mil 
 
Name, Rank, and Degree: MAJ Julianna Jayne, PhD, RD 
Title: Research Assistant 
Institution/Company: MND, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 508-233-5808 
Email: julianna.m.jayne.mil@mail.mil 
 
Name, Rank, and Degree: Michelle Saillant 
Title: Research Assistant 
Institution/Company: MND, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 508-233-5650 
Email: michelle.m.saillant.ctr@mail.mil 
 
Name, Rank, and Degree: Jillian Allen, MS, RD 
Title: Research Dietitian, ORISE Fellow 
Institution/Company: MND, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 508-233-4305 
Email: jillian.t.allen.ctr@mail.mil 
 
 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 8 of 59 
 
Name, Rank, and Degree: SGT Heather Hansen, BS 
Title: Research Assistant 
Institution/Company: BBMD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 480-489-7737 
Email: heather.m.hansen27.mil@mail.mil 
 
Name, Rank, and Degree: SPC David Galloway 
Title: Research Assistant 
Institution/Company: MPD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 210-420-3130 
Email: david.j.galloway4.mil@mail.mil 
 
Name, Rank, and Degree: SPC Joseph Bistolfi 
Title: Research Assistant 
Institution/Company: MPD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 731-616-5677 
Email: joseph.p.bistolfi.mil@mail.mil 
 
Name, Rank, and Degree: SPC Hector Figueroa 
Title: Research Assistant 
Institution/Company: BBMD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 404-966-4922 
Email: hector.r.figueroa21.mil@mail.mil 
 
Name, Rank, and Degree: SPC Kristine Chiusano 
Title: Research Assistant 
Institution/Company: MND, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 909-575-9674 
Email: kristine.k.chiusano.mil@mail.mil 
 
Name, Rank, and Degree: Karleigh Bradbury 
Title: Research Assistant 
Institution/Company: TMMD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 508-233-4977 
Email: karleigh.l.bradbury.civ@mail.mil 
 
Name, Rank, and Degree: Adam Luippold 
Title: Research Assistant 
Institution/Company: TMMD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 508-233-4977 
Email: adam.j.luippold.civ@mail.mil 
 
 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 9 of 59 
 
Name, Rank, and Degree: Beau Yurkevicius 
Title: Research Assistant 
Institution/Company: TMMD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 508-233-5950 
Email: beau.r.yurkevicius.ctr@mail.mil 
 
 
9 
Research Monitor 
 
Name, Rank, and Degree: MAJ Robin Cushing, DrPH, PA-C 
10 
Title: Medical Director 
11 
Institution/Company: OMSO, USARIEM 
12 
Address: 10 General Greene Ave, Bldg 86, Natick MA 01760 
13 
Phone Number: 508-233-5128 
14 
Email: robin.e.cushing.mil@mail.mil 
15 
 
16 
 
Ombudsmen  
  
 
Name, Rank, and Degree:  Katelyn Guerriere, MS 
Title:  Research Physiologist 
Institution/Company:  MPD, USARIEM 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number: 508-233-5619 
Email:  Katelyn.i.guerriere.civ@mail.mil 
 
Name, Rank, and Degree: Caitlin Haven, MS 
Title: Research Statistician 
Institution/Company: MPD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick MA 01760 
Phone Number: 508-233-4853 
Email: Caitlin.c.haven.civ@mail.mil 
 
Name, Rank, and Degree: Matthew Bartlett, BS 
Title: ORISE Research Fellow 
Institution/Company: MPD, USARIEM 
Address: 10 General Greene Ave, Bldg 42, Natick MA 01760 
Phone Number: 781-606-1819 
Email: paul.m.bartlett5.ctr@mail.mil 
 
17 
 
18 
A2.  ROLES AND RESPONSIBILITIES 
19 
      
20 
A2.1 Key Research Personnel 
21 
 
22 
Name(s):  Stefan M Pasiakos 
23 
Research Role:  Principal Investigator 
24 
Study Responsibilities:  The Principal Investigator is responsible for the safe and scientifically 
25 
sound conduct of the study.  He will oversee all aspects of the study, ensure safety and ethical 
26 
treatment of volunteers, maintain required documentation for the study and obtain required 
27 
approvals, and will have primary responsibility for data analysis, interpretation, and publication.  
28 
Dr. Pasiakos will be involved in volunteer briefing, obtaining informed consent, data collection, 
29 
performing muscle biopsies, catheterization, phlebotomy, exercise testing, and interventions.   
30 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 10 of 59 
 
 
31 
Name(s):  James P McClung, Stephen R Hennigar, Lee M Margolis, J Philip Karl, Harris R 
32 
Lieberman, Nicholas D Barringer, and Tracey J Smith, Jessica A Gwin, Alyssa N Varanoske 
33 
Research Role:  Associate Investigators 
34 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, 
35 
hypotheses, experimental approach, and design. Assist PI with volunteer briefing, obtaining 
36 
informed consent, exercise testing and interventions, oversight of daily volunteer monitoring, 
37 
data collection, management, analysis, and manuscript preparation.  Dr. Lee Margolis will also 
38 
perform exercise testing, muscle biopsies, catheterization, and phlebotomy. Dr. Gwin will also 
39 
perform exercise testing, muscle biopsies, catheterization and phlebotomy. MAJ Nicholas 
40 
Barringer and Alyssa Varanoske will additionally assist with exercise testing, DEXA and 
41 
phlebotomy. Drs. Margolis, Karl, Barringer, and Smith will assist with diet instruction and dietary 
42 
assessment. Dr. Karl will also assist with SmartPill administration and analysis. 
43 
 
44 
Name(s): Jennifer Rood 
45 
Research Role:  Associate Investigator 
46 
Study Responsibilities:  Receipt and analysis of coded data. Dr. Rood will not interact or 
47 
intervene with subjects or their identifiable data or specimens. 
48 
 
49 
Names (s): Svein Martini, Olav Vikmoen, Hilde Teien 
50 
Research Role: Associate Investigators 
51 
Study Responsibilities: Assist with study design, logistics, daily volunteer monitoring, meal 
52 
preparation and administration of study diets to volunteers, data analysis, and report generation.  
53 
Mr. Martini, Dr. Vikmoen, and Ms. Teien will only observe and assist with monitoring of 
54 
credentialed USARIEM research procedures.  They will not be preforming any credentialed 
55 
procedure.  
56 
 
57 
Name(s):  Rasha Hammamieh, Graham Finlayson, John Caldwell, Oshin Vartanian 
58 
Research Role:  Consultant 
59 
Study Responsibilities:  Assist with study design, logistics, data analysis, and report preparation. 
60 
These individuals will not interact or intervene with subjects or their identifiable data or 
61 
specimens. 
62 
 
63 
Name(s):  Adrienne Hatch 
 
64 
Research Role:  Project Coordinator 
65 
Study Responsibilities:  Supervise, manage, and coordinate study logistics and biological data 
66 
collection. She will be involved with protocol development, menu development, volunteer 
67 
briefing, oversight, preparation and administering study diets to volunteers, diet instruction, 
68 
dietary assessment, oversight of daily volunteer monitoring, and study implementation. She will 
69 
actively participate in data collection to include phlebotomy, exercise testing, interventions, 
70 
SmartPill administration, and DEXA.  She will assist in management, analysis and interpretation 
71 
of data, as well as preparation of manuscripts and technical reports for publication 
72 
 
73 
A2.2. Others Involved in the Research, as applicable  
74 
 
 
75 
Name(s):  Claire Whitney, Heather Fagnant, Nicholes Armstrong, Jillian Allen 
 
76 
Research Role:  Research Dietitians 
77 
Study Responsibilities:  Menu development, preparation and administering study diets to 
78 
volunteers, diet instruction, dietary assessment and study implementation, daily volunteer 
79 
monitoring. Claire Whitney, Heather Fagnant, and Nicholes Armstrong will also assist with 
80 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 11 of 59 
 
DEXA. Claire Whitney will also actively participate in data collection to include phlebotomy, 
81 
exercise testing, and interventions.  Nicholes Armstrong will also assist with phlebotomy and 
82 
catheterization. Ms. Fagnant will also assist with SmartPill administration and analysis. 
83 
 
84 
Name(s):  Nancy Murphy, Christopher Carrigan 
85 
Research Role:  Biological Sample Coordinators 
86 
Study Responsibilities:  Supervision, management, daily volunteer monitoring, and coordination 
87 
of logistics, and biological data collection. They will be involved with protocol development and 
88 
study implementation.  Data collection responsibilities will involve sample processing, 
89 
management, and oversight, and DEXA. Christopher Carrigan will also assist with phlebotomy 
90 
and catheterization. 
91 
 
92 
Name(s):  Patrick Radcliffe, Emily Howard, Michelle Saillant  
93 
Research Role:  Research Assistants 
94 
Study Responsibilities:  Assist with data collection, daily volunteer monitoring, meal preparation 
95 
and administering study diets to volunteers, and biological sample processing.  Mr. Radcliffe will 
96 
also assist with SmartPill administration and analysis. 
97 
 
98 
Name(s):  Marques Wilson 
99 
Research Role:  Research Assistant 
100 
Study Responsibilities:  Assist with data collection, daily volunteer monitoring, logistics and 
101 
biological sample processing. Marques Wilson will also be involved in phlebotomy, 
102 
catheterization, DEXA, and exercise testing and intervention. 
103 
 
104 
Name(s):  SPC Chiusano, SGT Rousayne, SSG Mason, SPC Sarpong, SPC Sanchez, SPC 
105 
Pounds, SPC Dare, SGT Hansen, SPC Galloway, SPC Bistolfi, SPC Figueroa, and Anthony 
106 
Karis 
107 
Research Role:  Research Assistants 
108 
Study Responsibilities:  Assist with data collection, daily volunteer monitoring, meal preparation, 
109 
administering study diets to volunteers, phlebotomy, and biological sample processing. SSG 
110 
Mason will also assist with catheterization. Mr. Karis will also assist with SmartPill administration 
111 
and analysis. 
112 
 
113 
Name(s):  Lauren Thompson, Philip Niro 
114 
Research Role:  Research Assistants 
115 
Study Responsibilities:  Facilitation and oversight of the mood, cognitive performance, and 
116 
vigilance assessments; assist with daily volunteer monitoring. 
117 
 
118 
Name(s):  Susan McGraw, MAJ Jayne 
119 
Research Role:  Research Assistant 
120 
Study Responsibilities:  Assist with meal preparation, daily volunteer monitoring, and 
121 
administering study diets to volunteers. Susan McGraw will additionally assist with DEXA. 
122 
 
123 
Name(s):  Karleigh Bradbury, Adam Luippold, Beau Yurkevicius 
124 
Research Role:  Research Assistants 
125 
Study Responsibilities:  Assist with meal preparation, daily volunteer monitoring, administering 
126 
study diets to volunteers, exercise testing and intervention, and DEXA. 
127 
 
128 
Name(s):  MAJ Robin Cushing 
129 
Research Role:  Research Monitor  
130 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 12 of 59 
 
Study Responsibilities:  The research monitor shall review all unanticipated problems involving 
131 
risk to subjects or others, serious adverse events and all subject deaths associated with the 
132 
protocol and provide an unbiased written report of the event. 
133 
 
134 
Name(s):  Katelyn I. Guerriere, Caitlin Haven, Matthew Bartlett   
135 
Research Role:  Ombudsmen 
136 
Study Responsibilities:  Observe group briefings for military volunteers not in the Human 
137 
Research Volunteer program.  
138 
 
139 
 
140 
A3.  RESEARCH LOCATIONS    
141 
 
142 
USARIEM, Natick MA: USARIEM is a DoD research facility within the US Army Medical 
143 
Research and Development Command.  It is the Institute responsible for conducting basic and 
144 
applied research to determine the effects of exposure to environmental extremes, occupational 
145 
tasks, physical training, deployment, operational stress and nutritional factors on the health and 
146 
performance of military personnel.  The facility contains environmental chambers for controlling 
147 
temperature and humidity, an environmentally controlled hypobaric chamber, a water immersion 
148 
laboratory, as well as several dry and wet laboratories for animal and human experimentation.  
149 
The dry laboratories are capable of a broad range of experiments, including biomechanical 
150 
analysis, body composition, energy expenditure, and muscle strength and endurance.  The wet 
151 
laboratories include general clinical chemistry analyzers, as well as equipment for ELISA, RIA, 
152 
histology, and molecular biology assays.  Each investigator at the facility has a personal 
153 
computer with software for data management, analysis, presentation and report generation.  
154 
Staff computers are interfaced with a network server for easy, secure data handling and 
155 
transfer.  All testing (pre-study screening, baseline, and experimental testing) will take place at 
156 
USARIEM. 
157 
 
158 
 
159 
SECTION B:  RESEARCH METHODOLOGY 
160 
 
161 
B1.  ABSTRACT   
162 
 
163 
Endurance exercise elicits skeletal muscle and systemic inflammation, reflected in large part by 
164 
increases in circulating concentrations of the pleotropic cytokine, interleukin-6 (IL-6).  Circulating 
165 
IL-6 facilitates skeletal muscle tissue repair and serves as an energy sensor during prolonged 
166 
endurance exercise by upregulating glycogenolysis to maintain blood glucose.  The IL-6 
167 
response to endurance exercise is normally attenuated with adequate rest and recovery as 
168 
skeletal muscle adapts with training.  However, performing repeated bouts of prolonged and 
169 
unaccustomed, muscle damaging (i.e., eccentric loading) endurance exercise may be 
170 
detrimental to performance and limit the adaptive responses to exercise by diminishing the 
171 
absorption of key nutrients (i.e., iron). Warfighters are commonly exposed to such exercise 
172 
bouts during sustained training and combat operations (SUSOPS), the effects of which may be 
173 
exacerbated by negative energy balance.  We recently reported that IL-6 and hepcidin, a 
174 
hepatic-derived protein that arises in response to inflammation and limits iron absorption, 
175 
increased by approximately 245% and 33%, respectively in Norwegian Soldiers participating in 
176 
a short-term (96 h) Arctic SUSOPS [1].  Circulating hepcidin concentrations post-SUSOPS were 
177 
associated with total daily energy expenditure (r = 0.4), energy intake (r = -0.26), and energy 
178 
balance (r = -0.43), suggesting that nutritional interventions designed to increase energy intake 
179 
during SUSOPS may attenuate IL-6 and its untoward downstream effects by limiting the 
180 

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magnitude of energy deficit.  However, these data, which were derived from an uncontrolled 
181 
military field study, are not indicative of causality and changes in iron absorption.  Therefore, to 
182 
define the putative role of energy balance on IL-6 and its downstream effects, we will conduct a 
183 
controlled laboratory study that simulates the physiological stressors imposed during SUSOPS 
184 
to determine if the IL-6 response is exacerbated by underfeeding.  Fifteen male, weight-stable, 
185 
active duty military personnel (aged 18 – 39 years) will be recruited to participate in a 22 d, 
186 
longitudinal study.  The study is comprised of four sequential phases: 1) a 72 h SUSOPS, 2) a 7 
187 
day recovery period (Recovery 1), 3) a second, 72 h SUSOPS, and 4) a final, 7 d recovery 
188 
period (Recovery 2).  During SUSOPS, subjects will be randomized to consume either sufficient 
189 
food (combat rations) to maintain energy balance within ± 10% of estimated total daily energy 
190 
expenditure (SUSOPS BAL) or will be provided only enough food to match 45% of total daily 
191 
energy expenditure to elicit severe negative energy balance (SUSOPS NEG BAL). Testing 
192 
procedures and primary outcome measures include dual energy x-ray absorptiometry (DEXA), 
193 
maximal and sub-maximal aerobic exercise testing, load carriage exercise, controlled feeding, 
194 
stable isotope assessments of energy expenditure, fractional iron absorption and whole-body 
195 
protein turnover, percutaneous muscle biopsies to assess intramuscular IL-6, glycogen status, 
196 
and regulators of muscle remodeling and substrate metabolism, intestinal permeability, gut 
197 
microbiome composition and function, and blood sampling to assess circulating concentrations 
198 
of IL-6 and other biomarkers of inflammation, muscle damage, nutritional, metabolic stress, and 
199 
immune responses to SUSOPS BAL and NEG BAL.  Physical performance will be assessed 
200 
before and after each SUSOPS and during each recovery period using the vertical jump test.  
201 
Self-reported mood, sustained attention, and cognitive performance will be assessed twice daily 
202 
using the Profile of Mood States, the Psychomotor Vigilance Task, the Evaluation of Risk 
203 
Questionnaire, the Match-to-Sample task, the Grammatical Reasoning test, and the N-Back 
204 
task.  Overall vigilance and nighttime sleep quality will be assessed via the wrist-worn 
205 
USARIEM Vigilance Monitoring System, the Fatigue Science ReadiBandTM actigraph, Philips 
206 
Respironics Actiwatch® Spectrum Plus, or equivalent device. This design will test the 
207 
hypothesis that maintaining energy balance will attenuate the IL-6 response to SUSOPS, 
208 
improve iron absorption, and attenuate the potential negative effects of excessive inflammation 
209 
on whole-body metabolic homeostasis.  Risks include those associated with venous 
210 
catheterization, venipuncture, muscle biopsies, DEXA, exercise, and the discomfort of severe 
211 
underfeeding.            
212 
 
213 
B2.  BACKGROUND AND SIGNIFICANCE  
214 
 
215 
Physiology of the Inflammatory Response to Exercise  
216 
Prolonged strenuous exercise is characterized by a large increase in circulating concentrations 
217 
of IL-6 (Figure 1A) followed by increases in cytokine inhibitors, such as IL-1 receptor antagonist 
218 
(IL-1ra), and the anti-inflammatory cytokine IL-10 [2-4]. In fact, concentrations of IL-6 
219 
consistently increase more than any other cytokine during an exercise bout [5]. The immediate 
220 
increase in circulating concentrations of IL-6 with exercise is mediated by the transcriptional 
221 
upregulation and release of IL-6 by contracting skeletal muscle fibers [6, 7]. Although many 
222 
cytokines are expressed in skeletal muscle (termed ‘myokines’) after prolonged exercise [8], IL-
223 
6 is the only myokine known to be released from skeletal muscle in appreciable concentrations. 
224 
IL-6 synthesized and released by contracting skeletal muscle during prolonged exercise is 
225 
thought to exert metabolic effects and is dependent on muscle glycogen availability [6, 7, 9] 
226 
(Figure 1B). For example, reduction in intramuscular glycogen prior to exercise increases 
227 
transcription of IL-6 mRNA in skeletal muscle and plasma IL-6 concentrations [6, 7], an effect 
228 
which may be mediated through activation of AMP-activated protein kinase [10] and p38 
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mitogen activated protein kinase [6]. Moreover, infusion of humans with recombinant human IL-
230 
6 (rhIL-6) induces a dose-dependent rise in blood glucose concentrations [11] and increases in 
231 
glucose disposal [12, 13], indicating that IL-6 may signal for hepatic glucose release to maintain 
232 
blood glucose during exercise. In addition to increasing hepatic glycogenolysis and 
233 
gluconeogenesis, rhIL-6 enhances lipolysis and fatty acid oxidation in adipocytes [14, 15], 
234 
perhaps to provide free fatty acids and energy when glycogen stores are low. 
235 
Another source of IL-6 is immune cells. During recovery from exercise, microstructural damage 
236 
as a result of microtrauma to contractile elements and connective tissue within muscle tissue 
237 
signal immune cell infiltration and secretion of cytokines (tumor necrosis factor (TNF)-α, IL-1β, 
238 
IL-6, IL-1ra, and IL-10) to heal the tissue. The rapid release of TNF-α and IL-1β stimulate the 
239 
production of IL-6, which in turn, is thought to be the central mediator in activating the acute-
240 
phase response and the systemic release of hepatocyte-derived acute phase proteins [16]. 
241 
These are likely delayed or secondary to the metabolic effects of IL-6 during exercise (i.e., 
242 
during recovery), as peak circulating concentrations of muscle-derived IL-6 occur immediately 
243 
following exercise and are acute, whereas IL-6 produced by immune cells occurs after this 
244 
peak, is of lower magnitude, and may remain elevated for an extended period of time to repair 
245 
the damaged tissue [5, 17, 18] (Figure 1A).  
246 
247 
Figure 1. Plasma IL-6 response to exercise and potential nutritional interventions. (A) IL-6 is released into 
248 
circulation by contracting skeletal muscle (i.e., ‘muscle-derived IL-6’) during prolonged, strenuous 
249 
exercise. This produces a large peak in circulating concentrations of IL-6 immediately after the completion 
250 
of exercise followed by a rapid decline to baseline concentrations. If tissue injury occurs with exercise 
251 
(e.g., strenuous eccentric exercise), immune cells infiltrate the muscle and release IL-6 to signal the 
252 
acute-phase response and tissue repair (i.e., ‘immune cell-derived IL-6’). This produces a rise in 
253 
circulating concentrations of IL-6 that is lower in magnitude, but more sustained than concentrations of IL-
254 
6 in circulating following exercise. Sufficient calories to match the energy requirement prior to exercise 
255 
and carbohydrate supplementation during prolonged, strenuous exercise may be an effective strategy to 
256 
reduce increases in muscle-derived IL-6 immediately following exercise. (B) Glycogen stores are depleted 
257 
with prolonged, strenuous exercise. Reductions in muscle glycogen signal for the transcriptional 
258 
upregulation of IL-6. IL-6 is released from contracting muscle, resulting in a rise in circulating levels of IL-
259 
6. IL-6 signals for hepatic glycogenolysis and gluconeogenesis. Prolonged exercise also signals for the 
260 
upregulation of IL-6 in adipose tissue and an increase in lipolysis. Circulating fatty acids and glucose and 
261 
fatty acids released from lipolysis of intramuscular triglycerides are transported to muscle mitochondria for 
262 
oxidation, resulting in increased ATP and energy for the exercising muscle. Dietary carbohydrate and lipid 
263 
increase available glucose and free fatty acids and stores of glycogen and triglycerides, thereby sparing 
264 
glycogen. ATP, adenosine triphosphate; FFA, free fatty acids; IL-6, interleukin-6 (figures adapted from 
265 
Hennigar et al. [19]). 
266 
Energy
Rest
Exercise
Recovery
Circulating [IL-6]
Muscle-derived IL-6
Immune cell-derived IL-6
Carbohydrates
Time 
LIVER
SKELETAL MUSCLE
Glycogen
Exercise
IL-6
IL-6
CIRCULATION
FFA
ATP
ADIPOSE
IL-6
Glucose
Gluconeogenesis
Glycogenolysis
Lipolysis
Dietary carbohydrates
Glucose & glycogen reserves
Dietary lipids
FFA & triglycerides
Lipolysis
FFA
A.
B.

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Factors that affect the source and magnitude of IL-6 released from muscle and immune cells in 
267 
response to exercise include mode and location of muscle mass, frequency, duration, intensity, 
268 
and training status [reviewed in [5, 13, 17, 18]]. In general, eccentric and strenuous exercise 
269 
involving multiple muscle groups for prolonged durations produces the greatest increase in IL-6 
270 
as a cytokine and myokine, respectively. Although skeletal muscle damage is not required for 
271 
the release of IL-6 from muscle during and immediately following exercise [5], it is important to 
272 
note that the release of muscle- and immune cell-derived IL-6 is not mutually exclusive. Higher 
273 
concentrations of muscle-derived IL-6 during and immediately following exercise generally 
274 
indicate greater muscle damage, leading to an increase in immune cell infiltration and the time 
275 
required for tissue repair and regeneration.  
276 
Potential Detriments of Inflammation on Iron Absorption during Sustained Operations 
277 
The release of IL-6 during exercise has been described as a double-edged sword [20], as IL-6 
278 
has the potential for both beneficial and detrimental effects. Beneficial adaptations resulting 
279 
from exercise occur when the stressor dose and the exercise bout are within a specific range 
280 
and followed by a rest period [21], whereas detrimental effects may occur when the dose or 
281 
exercise bout is excessive and not followed by periods of adequate rest and nutrition. For 
282 
example, repeated bouts of exercise on the same day or over the course of two days elevates 
283 
circulating concentrations of immune cells and induces a more pronounced increase in plasma 
284 
IL-6 as compared to a single bout of exercise [22, 23]. Further, repeated bouts of exercise 
285 
without adequate rest and nutrition may result in decreased performance, particularly if muscle 
286 
glycogen concentrations are low prior to exercise (e.g., fasted state, inadequate recovery, etc.) 
287 
or if the duration of exercise is too long to maintain glycogen stores.  
288 
Importantly, sustained elevations in IL-6 may result in a decline in nutrient absorption, 
289 
particularly the nutritionally essential mineral iron. Circulating levels of iron increase immediately 
290 
following exercise and decline with recovery [24]. Inflammation, specifically the release of IL-6, 
291 
is known to contribute to the decline in iron (termed the ‘hypoferremia of inflammation’). For 
292 
example, infusion of humans or animals with IL-6 reduces circulating concentrations of iron 
293 
>50% [25, 26]. The mechanism for the hypoferremia of inflammation is well established and 
294 
involves IL-6-induced increases in the hepatic, acute-phase protein hepcidin [26]. Hepcidin is 
295 
thought to bind to Cys326 in the extracellular domain of the iron exporter ferroportin [27], resulting 
296 
in the phosphorylation and subsequence ubiquitination of ferroportin [28]. The hepcidin-
297 
ferroportin complex is then endocytosed and degraded in the lysosome [29], thereby inhibiting 
298 
iron efflux into circulation. 
299 
Human and rodent models suggest that IL-6 may contribute to the exercise-induced increase in 
300 
hepcidin, as rises in hepcidin are subsequent to the post-exercise increase in muscle-derived 
301 
IL-6 [19]. Work from our laboratory indicates declines in iron status during military training [30, 
302 
31], concomitant with increased inflammatory biomarkers, including IL-6 and hepcidin [32, 33]. 
303 
As poor iron status is associated with a decrease in cognitive and physical performance [31] 
304 
these data indicate that prolonged or repeated IL-6-mediated increases in hepcidin during 
305 
training may be of concern if resulting in diminished iron status. 
306 
Nutritional Countermeasures to Inflammation: Role of Substrate Availability and Energy 
307 
Balance 
308 
Sparing muscle glycogen and maximizing recovery is essential for optimal performance during 
309 
exercise and in conferring the adaptive benefits of repeated exercise bouts with recovery (i.e., 
310 

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training). Because IL-6 is thought to respond to the energy available to the exercising muscle, 
311 
various nutritional interventions have been studied with the goal of mitigating increases in 
312 
circulating concentrations of IL-6 [19]. Collectively, these studies suggest that diets providing 
313 
adequate energy and that are high in carbohydrate (>60%) prior to exercise may attenuate the 
314 
depletion of glycogen stores and the resulting muscle-derived IL-6 response following exercise. 
315 
This is consistent with the notion that IL-6 release from contracting skeletal muscle is related to 
316 
pre-exercise glycogen availability [9] and that circulating concentrations of IL-6 post-exercise 
317 
are negatively correlated with skeletal muscle glycogen concentrations [34] such that the 
318 
duration and intensity of the exercise must be great enough to decrease skeletal muscle 
319 
glycogen concentrations for diet to have an effect.  
320 
Sustained Operations, Energy Balance and Inflammation: Observations from Military 
321 
Field Studies 
322 
Military personnel experience periods of negative energy balance resulting from increased 
323 
energy expenditure, limited dietary intake, or combined effects of both during sustained combat 
324 
and training operations (SUSOPS).   Warfighters engaged in various SUSOPS (i.e., 1-7 d 
325 
operations, separated by minimal rest (1-3 d), and repeated for ≥ a month) expend an average 
326 
of 4600 kcal/d [35, 36].  In some cases, energy expenditure may exceed these levels; total 
327 
energy expenditures for Marines engaged in mountain warfare training exceed 7000 kcal/d [37].  
328 
During military training and operations, energy supply and intake may be insufficient to maintain 
329 
energy balance, thus resulting in extreme energy deficits [35, 38].  For example, mean energy 
330 
expenditure during a 54-hr field training exercise was 6100 kcal/d for men, yet energy intake did 
331 
not exceed 1500 kcal/d, resulting in a significant loss of total body mass [39].  Prolonged or 
332 
repeat exposure to periods of negative energy balance can diminish physical performance, and 
333 
may increase the risk of injury.  
334 
Military training and operations have been associated with inflammation and degraded 
335 
nutritional status.  For example, short-term (7 d) military training results in significant elevations 
336 
in IL-6 and hepcidin [33].  Longer-term military training, such as basic combat training (7-10 wk), 
337 
results in diminished iron status, which may be due to the physiologic response to repeated 
338 
exposure to IL-6 [30-32].  A number of studies have investigated the effect of macronutrients, 
339 
including carbohydrates, on the inflammatory and immune response to physical activity [40, 41], 
340 
although these studies have not carefully controlled for energy intake.  The relationship between 
341 
energy balance and the inflammatory response, particularly the hepcidin response, is not well 
342 
described.  Previous studies from our laboratory show that  IL-6 and hepcidin increased by 
343 
approximately 245% (Figure 2A) and 33% (Figure 2B), respectively in Norwegian Soldiers 
344 
participating in a short-term 96-h Arctic SUSOPS that produced high energy expenditures 
345 
(~6000 kcal/d) and severe energy deficits (~50% total energy expenditure) [1].  Circulating 
346 
hepcidin concentrations post-SUSOPS were associated with total daily energy expenditure (r = 
347 
0.4), energy intake (r = -0.26), and energy balance (r = -0.43, Figure 2C) [1].  Collectively, these 
348 

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data indicate that interventions aiming at maintaining energy balance during periods of high 
349 
energy expenditure should be considered in efforts to attenuate the inflammatory response 
350 
associated with energy deprivation and military training.  Although observational studies have 
351 
uncovered statistical associations between energy balance and physiological outcomes, 
352 
intervention studies have not directly tested the hypothesis that preventing energy deficit 
353 
attenuates the inflammatory response, particularly the hepcidin response, to arduous physical 
354 
activity. 
355 
Figure 2. Circulating IL-6 (A) and hepcidin (B) concentrations before (PRE) and after (POST) completing 
356 
a 96 h Arctic SUSOPS, and the association between energy balance and post-SUSOPS hepcidin 
357 
concentrations (C).  *Different from PRE, P < 0.05 (data are mean ± SD). Figures adapted from Pasiakos 
358 
et al. [1].  
359 
Secondary Objectives 
360 
 
361 
Military Operational Stress, Intestinal Permeability, and Gut Microbiota Composition 
362 
 
363 
Decreased gut barrier integrity has been reported following exposure to physical and 
364 
psychological stressors such as those commonly experienced during military training [42-44].  
365 
The resulting increase in intestinal permeability can facilitate translocation of antigens from the 
366 
gut into systemic circulation thereby inducing inflammation that can worsen gut barrier 
367 
dysfunction [43].  A critical mediator of gut barrier integrity is the composition and activity of the 
368 
bacteria residing in the human gut, the gut microbiota [45].  Our laboratory recently reported a 
369 
positive association between changes in serum IL-6 concentrations and changes in intestinal 
370 
permeability in Norwegian Soldiers participating in a 96-h Arctic SUSOPS characterized by 
371 
severe energy deficit [44].  Changes in intestinal permeability and inflammation coincided with 
372 
pronounced shifts in gut microbiota composition and activity, and changes in intestinal 
373 
permeability were associated with both the composition of the microbiota before SUSOPS and 
374 
changes in microbiota activity during SUSOPS [44].  Severe energy deficit alone has been 
375 
shown to adversely affect gut barrier integrity, and gut microbiota composition and activity [46, 
376 
47].  The gap in knowledge is to what extent decrements in intestinal permeability and the gut 
377 
microbiome can be mitigated by maintaining energy balance during SUSOPS.  We will address 
378 
this gap by measuring intestinal permeability and gut microbiome composition and function. 
379 
 
380 
A primary finding from our Norwegian study was that of an increase in gut microbiota diversity 
381 
which was associated with increases in intestinal permeability during SUSOPS.  Greater 
382 
diversity in the microbiota is generally considered a marker of a healthy and more resilient gut 
383 
microbiota, and diversity has been reported to decrease in response to various stressors in 
384 
animal models [48, 49].  It was recently shown that longer intestinal transit time is associated 
385 
with increased gut microbiota diversity and changes in the relative abundance of multiple taxa 
386 
[50], implicating transit time as an important confounder in studies assessing the gut microbiota, 
387 
especially when studying effects of conditions known to impact intestinal transit.  Exercise [51], 
388 
underfeeding [52], and psychological and physical stress [53] have been independently shown 
389 
to alter gastrointestinal motility.  The effects of SUSOPS on gastrointestinal transit time, and its 
390 
impact on gut microbiota composition, are undetermined.     
391 
 
392 
Military Operational Stress, Appetite Regulation and Eating Behavior 
393 
 
394 
Warfighters frequently endure substantial energy deficit during SUSOPS, even when enough 
395 
food is provided to meet energy demands [54, 55].  This is counterintuitive because in most 
396 

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settings, energy deficit elicits a strong counter-regulatory response which stimulates appetite to 
397 
alleviate energy deficit and prevent weight loss.  Various psychological, logistical, and 
398 
environmental factors have all been cited as contributing to undereating during SUSOPS [54, 
399 
55], but whether appetite actually increases sufficiently to balance energy intake with elevated 
400 
energy expenditure is unclear.  The observation that endurance exercise transiently suppresses 
401 
appetite, increases appetite-suppressing hormone concentrations (e.g., peptide-YY (PYY), 
402 
glucagon-like peptide-1 (GLP-1), and pancreatic polypeptide (PP)), and depresses appetite-
403 
stimulating hormone concentrations (i.e., acylated ghrelin) suggests that the counter-regulatory 
404 
response to energy deficit could be blunted during SUSOPS [56-58].  However, to our 
405 
knowledge, this question has not been examined.  Moreover, changes in gastrointestinal transit 
406 
time have been associated with changes in appetite and appetite-mediating hormone secretion 
407 
[52, 59] suggesting that exercise- and/or energy deficit-mediated decreases in transit time could 
408 
contribute to appetite suppression during SUSOPS.  An improved understanding of how 
409 
appetite and biological factors regulating appetite (i.e., appetite-mediating hormones and 
410 
gastrointestinal transit rate) respond during SUSOPS is needed to optimize feeding strategies 
411 
within these environments, and to elucidate the contribution of these factors to eating behaviors 
412 
(e.g., food choice) that promote weight regain following SUSOPS.  As such, this study will 
413 
measure appetite, food preferences, and biological factors mediating appetite and food choice 
414 
before and after SUSOPS during both induced energy deficit and energy balance.  
415 
 
416 
A Systems Biology Approach for Characterizing Military Operational Stress 
417 
 
418 
Stress elicits systemic changes in gene and epigene expressions; behavior and performance; 
419 
metabolism, and immune function.  A comprehensive understanding of the spectrum of 
420 
pathways involved in regulating the stress response to military-relevant stress, and how those 
421 
pathways interact to influence health and behavior is needed to both identify biomarkers of 
422 
stress responses in Warfighters, and to elucidate targets for precision therapeutics aiming to 
423 
mitigate stress responses that may compromise Warfighter health and performance.  For 
424 
example, a systems biology approach interrogating the blood transcriptomic landscape was 
425 
recently used to identify 1400 transcripts that were differentially expressed in Soldiers before 
426 
and after Army Ranger Assessment and Selection [60], a notoriously grueling training program.   
427 
The analysis identified transcripts involved in the immune response as the most impacted 
428 
biological response to Ranger School, and elucidated pathways underlying impaired immune 
429 
function in this population.  This systems-biology approach will be expanded in this study by 
430 
integrating genomic and epigenomic (saliva), metabolomic (blood, feces, saliva), microbial 
431 
metagenomic (feces, saliva), and proteomic (saliva and blood) analyses.  In addition to 
432 
providing unique insight into the stress response in a military-relevant environment, this 
433 
approach will enable studying interactions among the microbiome and host physiology, the 
434 
“interactome”, to elucidate how the interactome responds to SUSOPS and the role of negative 
435 
energy balance in this response.     
436 
 
437 
B3.  MILITARY RELEVANCE    
438 
Warfighters are often exposed to physical and cognitive stress, which likely contribute to 
439 
documented declines in iron status, physical performance, and cognitive function during military 
440 
training [30, 31, 61]. Thus, it is critical to identify factors that contribute to the decline in iron 
441 
status and effective interventions to sustain and optimize Warfighter health and performance.  
442 
This highly-controlled study will delineate the effects of repeated bouts of strenuous activity 
443 
endured during SUSOPS on the IL-6 and hepcidin response, iron absorption and status, whole-
444 
body and skeletal muscle homeostasis, physical and cognitive performance, and determine if 
445 

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maintaining energy balance is an effective strategy to mitigate that response.  Data obtained 
446 
from this study may be used in the development of countermeasures to attenuate the 
447 
detrimental physiological effects of negative energy balance during strenuous military 
448 
operations.  Data obtained from this study will also be used to identify novel potential 
449 
biomarkers associated with the severity of stress responses to strenuous military operations.   
450 
 
451 
B4.  OBJECTIVES/SPECIFIC AIMS/RESEARCH QUESTIONS   
452 
 
453 
Objectives 
454 
 
455 
1. To determine the effects of a simulated 72-h SUSOPS on IL-6, hepcidin, and 
456 
fractional iron absorption.  
457 
 
458 
2. To determine whether energy balance and severe energy deficit modulate IL-6, 
459 
hepcidin, and fractional iron absorption during a simulated 72-h SUSOPS.  
460 
 
461 
Secondary Objectives 
462 
 
463 
1. To determine whether energy balance and severe energy deficit modulate 
464 
intramuscular inflammation, transcriptional modifications (e.g., microRNA and factors 
465 
involved with energy substrate metabolism), anabolic signaling, remodeling, and 
466 
whole-body (e.g., protein balance, immune function, etc.) adaptive responses to a 
467 
simulated 72-h SUSOPS.  
468 
 
469 
2. To determine to what extent maintaining energy balance mitigates decrements in 
470 
intestinal barrier integrity and in gut microbiome composition and function, and 
471 
influences gastrointestinal transit time during a simulated 72-h SUSOPS. 
472 
 
473 
3. To determine whether energy deficit increases appetite, and alters food preferences 
474 
and biological mediators of appetite (i.e., appetite-mediating hormones and 
475 
gastrointestinal transit time) during a simulated 72-h SUSOPS, and identify effects 
476 
on food choice following SUSOPS. 
477 
 
478 
4. To determine the effects of a simulated 72-h SUSOPS on physical performance, 
479 
mood, cognitive performance, vigilance assessed continuously, and nightly sleep 
480 
quality and determine whether energy deficiency exacerbates the effects of 
481 
SUSOPS. 
482 
 
483 
5. Characterize genomic and epigenomic stress markers in blood and saliva, 
484 
particularly markers interacting with the microbiome from gut and saliva, to build 
485 
networks that identify the “interactome” response associated with changes in energy 
486 
homeostasis during SUSOPS.  
487 
 
488 
Hypotheses 
489 
 
490 
1. SUSOPS will increase circulating concentrations of IL-6 and hepcidin and decrease 
491 
fractional iron absorption.  
492 
 
493 
2. Energy balance will attenuate circulating concentrations of IL-6 and hepcidin and 
494 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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improve fractional iron absorption compared to severe energy deficit. 
495 
 
496 
Secondary Hypotheses 
497 
 
498 
1. Energy balance will attenuate muscle glycogen depletion, intramuscular 
499 
inflammation, minimize upregulation in microRNA transcription, facilitate muscle 
500 
remodeling, and limit negative protein balance, and decrements in immune function.  
501 
 
502 
2. Energy balance will attenuate increases in intestinal permeability, increases in 
503 
gastrointestinal transit time, increases in markers of gut damage, and changes in gut 
504 
microbiota composition and function during SUSOPS. 
505 
 
506 
3. Energy deficit will increase appetite, change food preferences, increase circulating 
507 
concentrations of appetite-suppressing hormones, and suppress circulating 
508 
concentrations of appetite-stimulating hormones during SUSOPS, and alter food 
509 
choices following SUSOPS. 
510 
 
511 
4. SUSOPS will negatively impact physical performance, mood, cognition, and 
512 
vigilance, and SUSOPS also will alter the sleep quality of participants.  Negative 
513 
energy balance will exacerbate the effects of SUSOPS on these parameters. 
514 
 
515 
5. A systems biology approach integrating multi-omics data derived from fecal, blood, 
516 
and saliva will elucidate genetic targets within host-microbiome relationship 
517 
dynamics and biological pathways within the human host that are affected by 
518 
military-relevant stress and are associated with physical and cognitive performance, 
519 
and identify novel markers of physiological stress.  
520 
 
521 
B5.  RESEARCH PLAN   
522 
 
523 
B5.1 Research Design   
524 
 
525 
This randomized cross-over study will consist of four sequential phases (SUSOPS 1, Recovery 
526 
1, SUSOPS 2, and Recovery 2).  
527 
 
528 
B5.2 Research Subjects/Population(s) 
529 
 
530 
B5.2.1 Subject Population(s)   
531 
 
532 
Healthy, recreationally-active adult men, who are active duty military, will be recruited to 
533 
participate in this study. Male subjects were chosen because of known sex differences in 
534 
iron status and absorption and differences in their response to exercise, including the IL-
535 
6 response to exercise [62, 63].  Pending the outcomes from this study, future, more 
536 
targeted studies will be designed to address potential sex-based differences in 
537 
physiological responses to SUSOPS.   
538 
 
539 
B5.2.2 Number of Subjects, Records, and/or Specimens  
540 
 
541 
This protocol requires 15 subjects complete testing to provide 80% power to detect 
542 
significant differences in circulating hepcidin responses between SUSOPS 1 and 
543 
SUSOPS 2. Up to 60 subjects will be enrolled to account for attrition and screening 
544 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
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failures in order to get complete data on 15 subjects. See section B5.8.1 for details on 
545 
sample size calculations.  
546 
 
547 
B5.2.3 Inclusion Criteria 
548 
 
549 
 
Men who are active duty military, aged 18 – 39 years 
550 
 
Weight stable in the past 2 months (± 2.27 kg) 
551 
 
Healthy without evidence of chronic illness, medication use, or musculoskeletal injury 
552 
as determined by the USARIEM Office of Medical Support and Oversight (OMSO) 
553 
 
Recreationally active (2-4 days per week aerobic and/or resistance exercise) 
554 
 
Refrain from taking any pain-relievers (e.g., acetaminophen), nonsteroidal anti-
555 
inflammatory drugs (e.g., aspirin, Advil®, Aleve®, Naprosyn®), or any other aspirin-
556 
containing product for 10 days before starting and at least 5 days after completing 
557 
the study 
558 
 
Refrain from the use of alcohol and nicotine for the duration of the study  
559 
 
Willing to refrain from alcohol, smoking any nicotine product (includes e-cigarettes), 
560 
vaping, chewing tobacco, caffeine, and dietary supplement use, and from 
561 
consumption of probiotic-containing foods (e.g., yogurt, cottage cheese, sauerkraut, 
562 
etc.) and probiotic-containing supplements (e.g.,VSL#3, PRO-15, etc.) throughout 
563 
the entire study period (vitamin/mineral supplements cannot be taken for at least 2 
564 
weeks before starting the study) 
565 
 
Supervisor approval for non-HRV Active Duty Military working within the US Army 
566 
Natick Soldier Systems Center 
567 
 
Reports having a bowel movement at least as frequently as every-other-day 
568 
 
569 
 
570 
B5.2.4 Exclusion Criteria 
571 
 
572 
 
Musculoskeletal injuries that compromise exercise capability 
573 
 
Metabolic or cardiovascular abnormalities (e.g., kidney disease, diabetes, 
574 
cardiovascular disease, etc.)  
575 
 
History of any disease or abnormality of the gastrointestinal tract including (but not 
576 
limited to) diverticulosis, diverticulitis and inflammatory bowel disease, peptic ulcer 
577 
disease, Crohn’s disease, ulcerative colitis; or previous gastrointestinal surgery 
578 
 
Anemic (plasma ferritin < 40 µg/L, hemoglobin < 13 g/dL) and Sickle Cell 
579 
Anemia/Trait 
580 
 
C-reactive protein (CRP) > 5 mg/dL 
581 
 
Abnormal PT/PTT test or problems with blood clotting  
582 
 
History of complications with lidocaine  
583 
 
Evidence of any physical, mental, and/or medical conditions that would make the 
584 
proposed studies relatively more hazardous as determined by OMSO   
585 
 
Present condition of alcoholism or other substance abuse issues; use of anabolic 
586 
steroids  
587 
 
Blood donation within 4 months of beginning the study 
588 
 
Oral antibiotic use within 3 months of participation 
589 
 
Colonoscopy within 3 months of participation 
590 
 
Use of laxatives, stool softeners, or anti-diarrheal medications more than once/month 
591 
 
Currently using benzodiazepines, anti-depressants or anti-histamines 
592 
 
Pacemaker or other implanted electronic medical device 
593 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
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 
Are unwilling or unable to eat study diets and foods provided and/or follow exercise 
594 
prescriptions 
595 
 
596 
B5.3 Research Procedures   
597 
 
598 
 
599 
Experimental Design 
600 
After completing pre-study baseline testing [e.g., medical clearance, anthropometrics, diet and 
601 
activity records, resting metabolic rate (RMR), peak aerobic capacity (VO2peak)] and exercise and 
602 
performance familiarization trials, volunteers will complete a glycogen normalization protocol 
603 
and be provided a 48 h carbohydrate refeeding diet on days 1 and 2 to restore muscle glycogen 
604 
content pre-SUSOPS (Figure 3). On day 2, following an ≥ 8 h fast (0000 h), volunteers will 
605 
undergo the first of three fractional iron absorption studies (Iron  
606 
Study 1), during which time volunteers will ingest a stable isotopically labeled iron (54FeSO4) 
607 
drink followed by serial blood draws over the next 6 h. A fasting blood draw and percutaneous 
608 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
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muscle biopsy of the vastus lateralis will be collected on the morning of day 3. Volunteers will 
609 
also complete approximately 3-4 mood/cognitive-performance familiarization sessions and 1 
610 
baseline session before the SUSOPS testing period.  Immediately thereafter, volunteers will 
611 
complete a 72-h SUSOPS, and will be provided either sufficient food (combat rations) to 
612 
maintain energy balance within ± 10% of estimated total daily energy expenditure (SUSOPS 
613 
BAL) or  only enough food to match 45% of total daily energy expenditure to elicit severe 
614 
negative energy balance (SUSOPS NEG BAL). Volunteers will reside at the Doriot Climatic 
615 
Chambers during the SUSOPS beginning the evening of days 1 and 11 (volunteers will be 
616 
provided standardized meals), and ending after final data collection on days 6 and 16.  
617 
Throughout these periods, volunteers will wear the USARIEM Vigilance Monitoring System, the 
618 
Fatigue Science ReadiBandTM actigraph, Philips Respironics Actiwatch® Spectrum Plus, or 
619 
equivalent device on their non-dominant wrist.  The activities performed will be comprised of 
620 
load carriage and unloaded steady-state exercise and Warfighter operational tasks (e.g., 15 m 
621 
timed casualty evacuation drag of a 123 kg dummy, move under fire, prepare fighting positions) 
622 
to increase total daily energy expenditures.  Physical performance will be assessed before and 
623 
after each SUSOPS.  Mood, cognitive performance and overall vigilance will also be assessed.  
624 
Sleep will be restricted to four hours per night during SUSOPS to mimic recent field studies.  On 
625 
day 6, immediately following the final exercise bout and 8 h after dinner on day 5, a second 
626 
muscle biopsy will be collected (alternate leg from biopsy one) and volunteers will undergo Iron 
627 
Study 2 (57FeSO4). Days 7-12 will serve as a recovery period from SUSOPS 1 (Recovery 1).  
628 
Fasted blood draws will be collected on days 7-9 during Recovery 1, and a second baseline 
629 
mood/cognitive test session will occur during Recovery 1.  Starting on day 11, volunteers will 
630 
complete a second glycogen normalization and 48 h carbohydrate refeeding protocol.  On day 
631 
13, following an overnight fast, a blood draw and a third muscle biopsy (alternate leg from 
632 
biopsy two) will be collected.  Immediately thereafter, volunteers will complete the second 72 h 
633 
SUSOPS.  All testing, planned activity, and sleep restrictions will be the same as described for 
634 
SUSOPS I; however, volunteers will switch to either SUSOPS BAL or SUSOPS NEG BAL.  Iron 
635 
Study 3 (58FeSO4) and the fourth and final muscle biopsy (alternate leg from biopsy three) will 
636 
occur on day 16 immediately following the final exercise bout and 8 h after dinner on day 15.  
637 
Days 17-22will serve as Recovery 2 (7 d post-SUSOPS recovery blood draw will be collected on 
638 
day 22 to match the 7 d recovery from SUSOPS 1)  Diet records will be recorded on days 6-8, 
639 
and 16-18. 
640 
 
641 
SUSOPS BAL and SUSOPS NEG BAL 
642 
The SUSOPS will be comprised of a variety of Warfighter tasks, designed to elicit high energy 
643 
expenditures, muscle damage and fatigue, sleep deprivation, and decrements in physical and 
644 
cognitive performance as well as self-reported mood status.  Most importantly, the SUSOPS will 
645 
be designed to elicit a marked inflammatory response.  Physical activity/exercise will be 
646 
prescribed at levels to expend ~5000-6000 total kcal/d using the ACSM metabolic equations for 
647 
steady-state exercise [64] and the compendium of metabolic equivalents for physical activities 
648 
[65].  Total daily energy expenditure estimates will be consistent with our recent reports in 
649 
Norwegian Soldiers and matched between SUSOPS BAL and NEG BAL [55, 66].  Combat 
650 
rations (Meal Ready-to-Eat) will serve as the underlying diet during SUSOPS BAL and NEG 
651 
BAL (refer to SUSOPS Diet Intervention section for details).  Low-to-moderate intensity (30-65% 
652 
VO2peak) steady-state endurance-type exercise will be the primary exercise modality.    
653 
Volunteers will perform three prolonged steady-state exercise bouts per day.  All three exercise 
654 
sessions will be conducted outdoors on Natick Soldier Systems Center grounds (NSSC fitness 
655 
trail).  Two of the three exercise bouts will be ~60-180-min load carriage exercise sessions, 
656 
whereas the third will be unloaded. The total distance covered will be dictated by individual 
657 
exercise prescriptions, and the load carried will be ~32 kg [comprised of the basic uniform (~5.3 
658 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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v21OCT14 
 
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kg), weapon and tactical equipment (~11.2 kg), and rucksack (~15 kg)]. The loads carried are 
659 
consistent with infantry occupation standards. Volunteers will be permitted to consume water ad 
660 
libitum throughout each exercise trial to maintain hydration. During the remainder of each day, 
661 
volunteers will perform a number of Warfighter tasks [e.g., 15 m timed casualty evacuation drag 
662 
of a 123 kg dummy, move under fire (15, 6.6 m rushes with weapon and full combat load (32 kg) 
663 
for time, with 5 secs between rushes), prepare fighting positions (move 16, 18 kg sandbags 10 
664 
m while wearing full combat load minus a weapon (~26.5 kg)].  The Warfighter task measures 
665 
will be performed daily to increase energy expenditure by simulating some operational tasks. 
666 
Volunteers will perform at least two familiarization trials of each task to reduce injury risk during 
667 
the pre-study baseline period.  Mood, cognitive, and vigilance will also be assessed.  Self-
668 
reported mood and cognitive performance will be assessed twice per SUSOPS day in addition 
669 
to the sessions performed prior to SUSOPS 1 during the baseline testing phase of the protocol 
670 
and the session performed during Recovery 1.    Vigilance testing using the USARIEM Vigilance 
671 
Monitoring System will only be active during specific intervals of wake time. Sleep will be 
672 
restricted to 4 h per day to be consistent with previous USARIEM SUSOPS studies beginning 
673 
the evening of days 3 and 13(i.e., volunteers will not be permitted to sleep until 0100 the 
674 
morning of days 4 and 14) [67].  
675 
 
676 
Warfighter Tasks 
677 
Move under Fire: During this task, volunteers will wear an approximately 32 kg fighting load 
678 
[comprised of the basic uniform (~5.3 kg), weapon and tactical equipment (~11.2 kg), and 
679 
rucksack (~15 kg)]. They will begin the task in the prone position.  Upon command, volunteers 
680 
will sprint approximately 6 m to a marker and assume the predetermined position for that marker 
681 
(either the kneeling or prone position).  They will remain in this position for approximately 5 
682 
seconds.  Upon signal, volunteers will get up and sprint approximately 5 to 8 m to the next 
683 
marker and assume the predetermined position for that marker. This will be repeated until they 
684 
have covered a total of 100 m (15 rushes of ~6.6 m).  After starting in the prone position, the 
685 
next two positions will be kneeling, and then a prone position until the course is completed.  
686 
Time to complete the task will be recorded. Each testing session will take approximately 1-2 
687 
minutes.   
688 
 
689 
Casualty Evacuation Drag: Volunteers will drag a simulated casualty (approximately 123 kg) up 
690 
to 15 m as fast as possible in 60 sec, while wearing an approximately 32 kg fighting load 
691 
[comprised of the basic uniform (~5.3 kg), weapon and tactical equipment (~11.2 kg), and 
692 
rucksack (~15 kg)].  If the volunteer is unable to pull the casualty the full 15 m in 60 sec, the 
693 
distance the casualty was dragged will be measured to the nearest 0.25 m.  
694 
 
695 
Prepare Fighting Positions (Sandbag Carry): While wearing a fighting load minus the weapon 
696 
(approximately 26.5 kg), volunteers will lift and carry a total of 16 sandbags weighing 18 kg 
697 
each, carry them 10 m, and place them on the floor  in a 4 bag wide x 2 bag deep x 2 bag high 
698 
formation as quickly as possible.  The completion time will be recorded. The test is expected to 
699 
take approximately 3-5 minutes per volunteer. 
700 
 
701 
Diet and Total Daily Energy Expenditure Prescription during SUSOPS 
702 
Volunteers will complete a 3 d diet record and a 3 d activity log during baseline pre-study testing 
703 
(days -14-0) and according to instructions provided by a registered dietitian to characterize pre-
704 
study diet and exercise habits (see “Diet Record” and “Activity Log”).  To predict total daily 
705 
energy expenditure (TDEE), volunteers’ resting metabolic rate (RMR) will be measured during 
706 
the baseline pre-study testing period using standardized techniques and an indirect, open circuit 
707 
respiratory system (True Max 2400, ParvoMedics, Sandy, Utah, USA).  RMR will be multiplied 
708 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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v21OCT14 
 
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by a factor of 1.3 to estimate energy expenditures for activities of daily living.  Together, these 
709 
data will serve as the baseline total daily energy requirements for SUSOPS 1 and 2.   
710 
 
711 
To increase TDEE to approximately 5000-6000 kcal/d, volunteers will be prescribed low-to-
712 
moderate intensity (30-65% of pre-determined peak oxygen uptake, VO2peak and corresponding 
713 
heart rate) endurance-type exercise using the ACSM metabolic equations for steady-state 
714 
exercise [64] and the compendium of metabolic equivalents for physical activities (refer to 
715 
descriptions of Determination of Peak Oxygen Consumption and SUSOPS BAL and SUSOPS 
716 
NEG BAL below) [65].   
717 
 
718 
For example, an 85 kg individual with a baseline total daily energy requirement (RMR of 2000 
719 
kcal/d * 1.3) of 2600 kcal/d and a VO2peak of 45 mL/kg/min, would expend approximately 2468 
720 
kcal during two, 90-min load carriage exercise bouts combined [i.e., 85kg+32kg load = 117 kg * 
721 
(~50% of VO2peak is 6.7 METS *3.5 mL/kg/min)/1000) * (90 min of work * 5 kcal/L/min) = 1234 
722 
kcal/exercise bout], 600 kcal during a 60-min unloaded exercise session (calculations follow the 
723 
same format), and approximately 100 kcal/d performing the Warfighter tasks, for an estimated 
724 
TDEE of ~5768 kcal/d. TDEE prescriptions are individualized to each volunteer’s requirements 
725 
and will be held constant between SUSOPS 1 and 2. Exercise intensities, total duration 
726 
exercised, and TDEE will likely differ between volunteers.  Every attempt will be made to 
727 
maintain TDEE between 5000-6000 kcal/d across all volunteers; however, some individuals 
728 
may expend more and others less. 
729 
 
730 
Registered Dietitians will develop individualized daily menus for SUSOPS 1 and 2 using Food 
731 
Processor SQL (ESHA Research, Salem, OR, Version 10.14). The diets during each SUSOPS 
732 
period will be derived primarily from components of the US military Meals Ready-to-Eat (MRE) 
733 
rations to achieve appropriate macronutrient proportions.  The macronutrient distribution of the 
734 
MRE-based diets will not be manipulated.  Thus, volunteers will be consuming a diet providing 
735 
approximately 60% carbohydrate, 10-15% protein, and 25-30% fat. The macronutrient 
736 
distribution, as a % of total energy, will remain constant during SUSOPS NEG BAL by uniformly 
737 
reducing total energy intake across carbohydrate, protein, and fat.  This feeding paradigm will 
738 
most closely resemble field-feeding practices observed during recent USARIEM studies [55, 
739 
66].  The micronutrient content of the ration will not be altered.  However, to limit the potential 
740 
confounding effect of differing iron intakes across SUSOPS 1 and 2, volunteers will consume 
741 
supplemental iron sulfate mixed in water (Ferrous sulfate drops, RxChoice, available OTC) 
742 
during SUSOPS NEG BAL to match total iron consumed during SUSOPS BAL. This equates to 
743 
only ~4-10 mg supplemental iron and will be consumed with one meal each day to mimic iron 
744 
intake during SUSOPS BAL (iron content of SUSOPS BAL is approximately 20-25 mg/d). Water 
745 
will be allowed ad libitum and total amount consumed will be recorded by study team members. 
746 
During the 48 hours prior to each SUSOPS period (beginning on days 1 and 11, volunteers will 
747 
be fed meals derived of commercially available foods which will meet calculated energy 
748 
requirements to maintain their body weight.      
749 
 
750 
Volunteers will receive instructions from study dietitians on how to consume an ad libitum diet 
751 
with the same macronutrient distribution during the two recovery periods (see “Choosing Your 
752 
Foods During Recovery” document).  Food records will be maintained by study volunteers on 
753 
days 6-8 and 16-18.  Volunteers will meet with study dietitians to review these records. These 
754 
records will be used to determine how energy deficit during SUSOPS impacts food selection 
755 
during recovery.  Physical activity will be restricted during both recovery periods.   
756 
 
757 
Determination of Total Daily Energy Expenditure 
758 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
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Doubly labeled water (DLW) will be used to determine actual TDEE during SUSOPS 1 and 
759 
SUSOPS 2 and verify the accuracy of estimated TDEE.  DLW will be administered during the first 
760 
iron study on day 2 (~0000 h after an 8 h fast).  Immediately before drinking the DLW, volunteers 
761 
will provide a urine sample to determine the natural abundance of 18O and 2H.  They will not eat or 
762 
drink anything for 4 h (~0400 h) after consuming a total of 120 g DLW containing 10% H218O 
763 
(~0.285 g H218O·kg total body water [TBW]-1) and 99% 2H2O (~0.15 g 2H2O·kg TBW-1; Sigma-
764 
Aldrich, St. Louis, MO or similar company).  The DLW dose container will be rinsed with local tap or 
765 
bottled water, which the volunteer will drink.  Urine samples will then be collected approximately 4 h 
766 
and 6 h after the DLW dosing for initial TBW determinations to be made.  Volunteers will be free to 
767 
eat and drink after these urine samples.   
768 
 
769 
Two volunteers will be chosen at random to consume only locally available drinking water to control 
770 
for natural changes in 2H and 18O abundance (local water will be analyzed to determine isotopic 
771 
enrichments).  Rate of disappearance of 18O and 2H for volunteers dosed with DLW will be 
772 
corrected for mean changes in background enrichments based on controls.  Morning urine samples 
773 
will be collected daily during each SUSOPS period to determine elimination rates over time.  TBW 
774 
will be calculated by determining the regression line for the elimination of 2H and 18O and 
775 
extrapolated to a maximum enrichment. All urine samples will be collected and stored in 5ml tubes 
776 
and shipped to the Pennington Biomedical Research Center (PBRC) for analysis.   
777 
 
778 
Enrichments of 2H and 18O will be measured using isotope ratio mass spectroscopy (Finnigan Mat 
779 
252, Thermo Fisher Scientific, Waltham, MA or similar model).  The 2H and 18O isotope elimination 
780 
rates (kH and kO) will be calculated by linear regression using the isotopic disappearance rates 
781 
during each training phase. 
782 
 
783 
rCO2 = (N/2.078)(1.01kO – 1.04kH) – 0.0246rH2Of 
784 
 
785 
where N is TBW; kO and kH are 18O and 2H isotope disappearance rates respectively, and rH2Of  
786 
is the rate of fractionated evaporated water loss and is estimated to be 1.05 N * (1.01 kO – 1.04 
787 
kH).  Total daily energy expenditure will then be calculated using the energy equivalent of CO2 
788 
for a respiratory quotient of 0.86 [68].   
789 
 
790 
Anthropometric Data 
791 
Anthropometrics and body composition will be performed using standardized techniques and 
792 
equipment to characterize study volunteers, and evaluate responses to SUSOPS 1 and 2.  
793 
Height will be measured in duplicate to the nearest 0.1 cm using a stadiometer at baseline. 
794 
Body weight will be measured, nude (scale will be placed in a locked bathroom) and after an 
795 
overnight fast (≥ 8 h), using a calibrated digital scale to the nearest 0.1 kg at baseline (days -14-
796 
0) and then daily throughout the 22 d study to ensure weight maintenance and/or weight loss.   
797 
 
798 
Body composition will be determined on days 2, 6, 12, and 16 using a four-compartment model 
799 
derived from dual energy x-ray absorptiometry (DEXA, DPX-IQ, GE Lunar Corporation, 
800 
Madison, WI) and bio-electrical impedance (InBody 720, BIOSPACE, Korea) [69]. The DEXA 
801 
technique allows for the non-invasive assessment of soft tissue composition by region with a 
802 
precision of 1-3%. The volunteer will lay face-up on the DEXA densitometer table in shorts, t-
803 
shirts, and stocking feet, and will be asked to remain motionless for the 8-10 min scan. The 
804 
InBody measure takes less than a minute to complete, will be performed immediately before or 
805 
after the DEXA, with volunteers wearing the same clothing described for DEXA.  These data will 
806 
be used to calculate body water, protein (i.e., lean mass), mineral (i.e., bone), and fat mass.  
807 
 
808 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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Determination of Peak Oxygen Uptake 
809 
Peak oxygen uptake (VO2peak) will be determined using an indirect, open circuit respiratory 
810 
system (True Max 2400, ParvoMedics, Sandy, Utah, USA) on a cycle ergometer.  The value will 
811 
be used to better estimate exercise-induced energy expenditure (EIEE) during SUSOPS and to 
812 
determine the workloads necessary for the glycogen normalization protocol (see below).  
813 
Volunteers will be clothed in appropriate athletic attire and perform this assessment in a 
814 
temperature and humidity controlled room.  The volunteer will be allowed to warm-up by 
815 
pedaling at 70 W for 5 min.   At the start of testing, the volunteer will put on a nose clip and a 
816 
mouthpiece connected to a 2-way respiratory valve, which is attached to a head piece to hold it 
817 
in place.  Every minute, workload intensity will be progressively increased by 30 W until the 
818 
volunteer is fatigued or unable to maintain a pedaling rate that either maintains or increases O2 
819 
consumption.   Heart rate will be monitored using a heart-rate monitor (Polar Electro Inc, Oulu, 
820 
Finland) and recorded during the last 30 sec of each workload.  The test will be stopped 
821 
immediately if the subject reports angina-like symptoms, exertional syncope, shows signs of 
822 
poor perfusion (i.e., light-headedness, confusion, ataxia, pallor, cyanosis, nausea, or cold and 
823 
clammy skin), or if the testing equipment fails.   
824 
 
825 
Glycogen Normalization Protocol 
826 
To normalize muscle glycogen content and its potential influence on inflammation leading into 
827 
both SUSOPS periods [days 1 and 11 following an overnight fast (≥ 10 h)], participants will 
828 
perform a glycogen normalization protocol followed by a 2-d refeeding protocol. The glycogen 
829 
normalization protocol will be completed on a cycle ergometer. The intensity will be based on 
830 
VO2peak.  Volunteers will begin with a 5 min warm-up at 50% VO2peak before beginning the 
831 
protocol.  After a warm-up period, the cycle ergometer protocol is comprised of repeated periods 
832 
of 2 min of work at 80 ± 5% V̇ O2peak followed by 2 min of recovery at 50 ± 5% VO2peak.  The 
833 
protocol will last approximately 50-min (12 work:rest cycles). To ensure familiarity with the 
834 
testing procedures, volunteers will perform one practice session during the baseline pre-study 
835 
period.  Volunteers will be permitted to consume water ad libitum during the protocol.   
836 
 
837 
After completing the glycogen depletion protocol, volunteers will be fed a controlled diet 
838 
prescribed to maintain energy balance and provide at least 6.0 g carbohydrate ∙kg-1∙d-1 (~55-
839 
60% of total energy consumed) to ensure adequate glycogen repletion and homogeneous 
840 
glycogen levels within and across volunteers between SUSOPS phases (BAL and NEG BAL).  
841 
All food and beverages (except water) will be prepared and provided by study dietitians and 
842 
consist largely of military combat ration and supplemental food items.   
843 
 
844 
Determination of Fractional Iron Absorption 
845 
Volunteers will undergo three acute fractional iron absorption studies (Iron Studies 1, 2, and 3).  
846 
The studies will occur on days 2, 6, and 16 in the morning (~0000 h) after an 8 h fast.  The iron 
847 
studies on days 6 and 16 will occur ~3 h after completing the last exercise bout of the SUSOPS 
848 
to correspond to peak hepcidin responses to exercise.  An indwelling 18 gauge intravenous 
849 
catheter will be placed in the antecubital fossa (or distally) and a baseline blood sample will be 
850 
drawn before consuming the iron isotope (0 min).  Volunteers will then consume a 300 mL drink 
851 
containing 3.8 mg iron (representative of dietary iron in an iron-rich meal) as isotopically labeled  
852 
54FeSO4, 57FeSO4, or 58FeSO4 (Trace Sciences International). Venous blood samples will be 
853 
collected at 20, 40, 60, 120, 240, and 360 min-post iron loading [70] to assess the effects of a 
854 
transient exercise-induced increase in IL-6 (and hepcidin) on the appearance of absorbed iron 
855 
in blood.   
856 
 
857 
Percutaneous Muscle Biopsy of the Vastus Lateralis  
858 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
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Percutaneous muscle biopsies will be obtained from the vastus lateralis using a 5 mm 
859 
Bergstrom needle with manual suction while the volunteer is under local anesthesia (1% 
860 
lidocaine) according to the approved USARIEM SOP [71, 72].  The biopsy procedures will be 
861 
performed after a ≥ 8 h fast immediately before starting and after completing each SUSOPS 
862 
period (days 3, 6, 13, and 16).  The muscle biopsies collected on days 6 and 16 will occur within 
863 
30 min of completing the final exercise bout before starting iron tracer studies 3 h post-exercise 
864 
(refer to Determination of Fractional Iron Absorption). The muscle samples obtained will be 
865 
analyzed for muscle glycogen content, intramuscular markers of inflammation, proteolytic, 
866 
energy-sensing, and anabolic cell signaling. Volunteers will undergo four total biopsies.  Each 
867 
biopsy will require a new incision and the biopsied leg will alternate between days.    
868 
 
869 
Whole-Body Protein Utilization 
870 
Whole-body protein turnover (15N-alanine) will be assessed before (days 2 and 12) and on the 
871 
last day of each SUSOPS period (days 5 and 15), as previously reported in several studies from 
872 
this laboratory [55, 66, 73, 74].  Total nitrogen, ammonia, and urea (15N-nitrogen, 15N- ammonia, 
873 
and 15N-urea) enrichments will be used to measure whole-body protein turnover [75].  After 
874 
providing a urine sample to determine background 15N enrichments, volunteers will consume a 
875 
single dose of 15N-alanine (300 mg·d-1, Cambridge Isotope Laboratories, Andover, MA) and 
876 
collect their urine for the next 24 h.  Total nitrogen intake will be determined from ration dietary 
877 
analysis.    
878 
 
879 
Urine samples will be frozen and shipped to Metabolic Solutions Inc. (Nashua, NH) for isotopic 
880 
analysis using a fee-for-service contract.  The 15N enrichment of urinary ammonia and N (ratio 
881 
of tracer to tracee, t:t) will be determined using isotope ratio mass spectroscopy.  The t:t ratio for 
882 
the cumulative sample will be corrected for the background 15N-ammonia enrichment.  Nitrogen 
883 
intake (I) during the 24 h period will be determined by ration analysis.  Nitrogen flux (Q), protein 
884 
synthesis (PS), protein breakdown (PB), and net protein balance (NET) will be calculated 
885 
according to Stein et al.[76].   
886 
 
887 
Determining Lower-Body Performance 
888 
Physical performance (i.e., lower-body power) will be assessed before and after each SUSOPS 
889 
and during each recovery period on days 3, 5, 7, 9, 13, 15, 17, 19using the vertical jump test 
890 
(Vertec™ device). Volunteers will place their feet at shoulder width with their knees slightly bent 
891 
while standing on a flat, clean surface.  On command, volunteers will perform an arm swing and 
892 
a countermovement to a self-selected depth and jump with maximal effort.  Volunteers will tap 
893 
the fins of the Vertec™ at peak jump height. Volunteers will receive three attempts separated by 
894 
one minute of recovery. Vertical displacement (jump height) will be calculated as the difference 
895 
between maximal jump height and reach height and peak power will be calculated using the 
896 
following equation [77].  
897 
 
898 
Peak Power (Watts) = [60.7 x jump height (cm)] + [45.3 x body mass (kg)] - 2055 
899 
 
900 
Volunteers will receive detailed instruction, demonstration by study staff, and complete two 
901 
familiarization trials during the pre-study period.  All experimental conditions will be held 
902 
constant between testing days. 
903 
 
904 
Eating behavior, appetite and food preferences and cravings 
905 
Volunteers will rate self-perceived appetite using visual analog scales (see “VAS” document).  
906 
The VAS will be administered before and after meals, and before and after morning exercise on 
907 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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study days -1 (may be adjusted ± 2d if needed), 3-5, and 13-15, (note on day -1 the VAS will be 
908 
administered at the same time of day as the morning exercise sessions on days 3-5, and 13-
909 
15).  Visual analog scales will be administered using paper and pencil, or via electronic means 
910 
(e.g., tablet or laptop).  During study day -1, participants will remain in the lab under staff 
911 
supervision from breakfast through dinner (~0600-~1900).  All meals will be provided in 
912 
sufficient quantity to meet weight maintenance energy requirements, and participants will 
913 
remain sedentary throughout the day.  Appetite-mediating hormones (acyl ghrelin, leptin, GLP-
914 
1, PYY, PP, and insulin) and metabolic markers putatively involved in appetite regulation 
915 
(glucose, FFA, BHB) will be measured following a ≥8 hr fast on the mornings of study days 3, 5, 
916 
7, 9, 13, 15, 17, and 19 and following the morning ruck march on days 3, 5, 13 and 15. 
917 
   
918 
Food preferences will be measured using the Leeds Food Preference Questionnaire (LFPQ) 
919 
which will be administered before and after lunch on study days -1 (may be adjusted ± 2d if 
920 
needed), 5, and 15.  The LFPQ is a computerized platform that measures different components 
921 
of food preference and hedonics, and is administered on a computer [78].  For the tests, 32 
922 
pictures of individual food items that vary in 3 dimensions; protein (low and high), taste (sweet 
923 
and savory), and/or fat (low and high) will be selected from a validated database of common 
924 
foods. At least four pictures from each food type will be selected.  To measure explicit liking 
925 
(i.e., perceived hedonic impact of the food), the volunteer will be shown each picture in random 
926 
order and asked to respond to the question “how pleasant would you find the taste of this food 
927 
right now” using a visual analog scale. To measure explicit wanting (i.e, conscious desire to 
928 
consume a food), the volunteer will be shown each picture in random order and asked to 
929 
respond to the question “how much do you want to eat this food right now”, using a visual 
930 
analog scale. Mean ratings for each food category will be computed to determine explicit liking 
931 
and explicit wanting for each food type. Implicit wanting (i.e., automatic subconscious attraction 
932 
to a food) will be measured using forced choice methodology.  Pairs of food images from 
933 
separate food categories will be presented in randomized pairs on the computer screen. 
934 
Volunteers will be asked to as quickly as possible select the food that they most want to eat at 
935 
that moment. Both frequencies of selections within each food category and response time will 
936 
be recorded.  A familiarization session will be conducted before study day -1.  The LFPQ will be 
937 
will be purchased or used with permission and takes ~15min to complete 
938 
 
939 
Gut microbiome composition and activity 
940 
Volunteers will collect 5 separate fecal samples to determine the effects of SUSOPS with and 
941 
without energy deficit on gut microbiome composition and activity.  A single fecal sample will be 
942 
collected during baseline (days -1-0 (may be adjusted ± 2d if needed)), and study days 5-6, 9-
943 
10, 15-16, and 19-20. At each time point participants will be given a 48-h window to collect a 
944 
usable sample.  A usable sample is defined as being >15g wet weight, and having been 
945 
delivered to study staff as soon as possible and within 12 hr of defecation while being kept cold 
946 
but not frozen from the time of collection to delivery. If a participant does not provide a usable 
947 
sample within the timeframe noted above, the collection period will be extended until a usable 
948 
sample is produced. To collect fecal samples, all participants will be given pre-labeled 
949 
containers with covers and a plastic device to hold the container in the toilet. Participants will 
950 
defecate into the collection container which will then be given to study staff.  During free-living 
951 
phases of the study participants will be given plastic sealable bags, a cooler or insulated bag, 
952 
and ice packs to store and transport the samples (see “Fecal Collection Instructions”).  Gut 
953 
microbiota composition, function, and activity will be measured at the US Army Center For 
954 
Environmental Health Research (USACEHR), Ft Detrick using next generation shotgun-
955 
sequencing and non-targeted metabolomics. Additional aliquots will be archived by USARIEM at 
956 
-80○C and may be analyzed for other gut related outcomes at a later date.  
957 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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958 
Gastrointestinal permeability 
959 
A differential sugar absorption test will be used to provide a functional assessment of 
960 
gastrointestinal permeability [79]. For this test participants will consume 2 g sucralose and 2 g 
961 
erythritol dissolved in 180 mL water in the morning during study days -1 (may be adjusted ± 2d if 
962 
needed), 4, and 14. Sucralose and erythritol are sugar substitutes commonly used in a variety of 
963 
food products. Consumption of the solution will be conducted under staff supervision. Participants 
964 
will then collect urine produced over the subsequent 24 hr.  Urine aliquots will be taken after 5 hr 
965 
and 24 hr and frozen immediately. Urine sucralose and erythritol concentrations will be analyzed by 
966 
PBRC, and will provide a measure of small intestinal, large intestinal and whole-gut permeability 
967 
[79].  Coolers or insulated bags with ice packs will be used to keep urine cool and for transport of 
968 
samples outside of the lab (see “Fecal Collection Instructions”).   
969 
 
970 
Gastrointestinal transit time 
971 
Gastrointestinal transit time will be measured on days -1, 4 and 14 using the SmartPill 
972 
(Medtronic, Minneapolis, MN).  The SmartPill is an ingestible FDA-approved wireless motility 
973 
capsule similar in size to a multi-vitamin pill that transits the gastrointestinal tract while 
974 
transmitting data to a receiver kept near the body 
975 
[http://www.medtronic.com/covidien/products/motility-testing/smartpill-motility-testing-system#].  
976 
The device continuously measures gastrointestinal pH, core temperature, gastrointestinal 
977 
pressure, and gastrointestinal transit time for up to 5 d, and can isolate transit through the 
978 
stomach, small bowel, and colon [80].  One sterile SmartPill will be ingested immediately after 
979 
breakfast on days -1, 4, and 14 under staff supervision.  The pill is easy to swallow and 
980 
volunteers will be given ample instruction. Elimination of the pill from the gastrointestinal tract 
981 
will be confirmed by a temperature decrease when the capsule passes from the body into toilet 
982 
water.  If a pill stops transmitting before being eliminated from the body, a new pill may be 
983 
administered and fecal sample collections will be used to visually search for and confirm exit of 
984 
the pill that is no longer transmitting.  We expect most volunteers will pass the capsule within 
985 
24-48 hr based on data from Rao et al. [81] who reported median [IQR] whole gut transit time of 
986 
30 hr [22-46 hr] in 81 healthy adults.  Volunteers will wear a bracelet indicating the pill is inside 
987 
of them until elimination from the body is confirmed by study staff.  Transit time will be used in 
988 
interpretation of the gut microbiota, gastrointestinal permeability, and appetite results as 
989 
changes in transit time could impact all of these measures. A subset of volunteers will be 
990 
recruited randomly to participate in this procedure using a random number generator. If any 
991 
volunteer who is randomly selected reports difficulty swallowing large pills or any other 
992 
contraindication (see below), they will be replaced with a randomly selected alternate. 
993 
 
Contraindications: 
994 
 
History of gastric bezoar 
995 
 
Swallowing disorders; severe dysphagia to food or pills 
996 
 
Suspected or known strictures, fistulas, or physiological/mechanical GI obstruction 
997 
 
Implanted or portable electro-mechanical medical devices  
998 
 
Using proton pump inhibitors 
999 
 
Cannot stop use of the following medications within 48hr of procedure: histamine 
1000 
blockers, GI motility-altering medications, antiemetics & 5HT3 antagonists, macrolides, 
1001 
anticholinergics, 5HT4 partial agonists, antacids 
1002 
 
1003 
Gastrointestinal health log 
1004 
Frequency of bowel movements and subjective ratings of gastrointestinal symptoms (e.g., 
1005 
flatulence, constipation, loose stool) will be assessed during study days -1 (may be adjusted ± 2d if 
1006 
needed), 3, 6, 13, and 16, using modified versions of the Irritable Bowel Syndrome-Symptom 
1007 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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Severity Score Questionnaire [82] and the Gastrointestinal Quality of Life Index [83] (see 
1008 
“Gastrointestinal Health Log”). Both questionnaires are publicly available and take <5min to 
1009 
complete. 
1010 
 
1011 
Assessment of mucosal immunity  
1012 
Secretory IgA will be assessed from saliva samples at baseline and each morning of SUSOPS 
1013 
to evaluate mucosal immunity. Saliva samples will be collected using the non-invasive 
1014 
commercially-available Salimetrics polyester Oral Swab (SOS) technology (Salimetrics, CA, 
1015 
USA). Subjects will be asked to place one SOS under their tongue for 3 minutes before 
1016 
returning it to the provided vial. Participants will then place a second SOS under their tongue 
1017 
until the swab is saturated with saliva. This sampling technique will enable us to obtain the 
1018 
required amount of saliva to determine sIgA concentration and the salivary flow rate at each 
1019 
time point. Salivary SIgA is a well-documented biomarker of mucosal innate immunity, used to 
1020 
predict upper respiratory tract infections and to study the influence of stress and dehydration on 
1021 
immune function [84-86]. The collected saliva will be stored at -80̊C until analysis. Saliva 
1022 
osmolality will be determined using a freezing point depression osmometer, and samples will be 
1023 
analyzed simultaneously for sIgA concentration using a commercially-available ELISA.  
1024 
 
1025 
Self-Reported Mood, Cognitive Performance, and Vigilance Assessments 
1026 
Several mood and cognitive performance assessment sessions will be conducted during the pre-
1027 
SUSOPS period of testing so that volunteers become familiar with the test battery and their 
1028 
performance is stable. The last of these sessions will serve as the baseline data for statistical 
1029 
analyses.  During the simulated SUSOPS periods, a morning and evening mood/cognitive test 
1030 
session will be conducted on each day.  Ambulatory vigilance will be assessed via the wrist-worn 
1031 
USARIEM Vigilance Monitoring System.  The Fatigue Science ReadiBand™ actigraph, Philips 
1032 
Respironics Actiwatch® Spectrum Plus, or an equivalent device will record sleep data at night and 
1033 
daytime motor activity. (see “POMS” and “EVAR” documents). 
1034 
 
1035 
The Profile of Mood States (POMS): The Profile of Mood States (POMS) Questionnaire [87] is a 65-
1036 
item inventory of self-reported mood states that is sensitive to a wide variety of nutritional 
1037 
manipulations including undernutrition[88] and environmental factors including hypoxia [89], sleep 
1038 
loss, and sub-clinical doses of various drugs [90-92].  Participants rate each of 65 mood-related 
1039 
adjectives on a five-point scale, in response to the question, “How are you feeling right now?”  The 
1040 
adjectives factor into six mood sub-scales (tension/anxiety, depression/dejection, anger/hostility, 
1041 
vigor/activity, fatigue/inertia, and confusion/bewilderment.  The POMS will be used to assess the 
1042 
overall mood states of the participants in the present study.  The POMS Questionnaire takes less 
1043 
than 5 minutes and will be administered via computer software.  This questionnaire will be 
1044 
purchased and used with permission. 
1045 
 
1046 
Evaluation of Risks Scale (EVAR): The Evaluation of Risks (EVAR) Questionnaire measures 
1047 
willingness to take risks through participant responses to 24 items which they mark on a visual 
1048 
analog scale [93].  Each end of the line is anchored by descriptors such as “not at all” and “very 
1049 
much.” Respondents simply mark the point on the line that best describes their current feeling state. 
1050 
The scale yields five factors, including Self-control, Danger-seeking, Energy, Impulsiveness, and 
1051 
Invincibility, as well as a Total Risk-Taking Propensity score, which is derived by summing all 24 
1052 
items. These items are internally consistent, yielding a coefficient a of 0.78 [94]. The scale has been 
1053 
shown to differentiate individuals who routinely engage in risky behavior, and it also correlates with 
1054 
measures of sensation-seeking and other risk-related traits [93, 94]. The EVAR takes less than 5 
1055 
minutes to complete.  This questionnaire is publicly available. 
1056 
 
1057 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
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The Psychomotor Vigilance Test (PVT): The Psychomotor Vigilance Test (PVT) measures simple 
1058 
visual reaction time which is particularly sensitive to the vigilance decrements associated with sleep 
1059 
restriction or disruption [95]. A series of stimuli are presented at random intervals on a screen and 
1060 
the subject must respond as rapidly as possible when a stimulus appears. The subject hits either 
1061 
the left or right arrow keys to respond to the stimulus. Parameters recorded include reaction time, 
1062 
false alarms, and number of lapses (long duration responses). PVT performance lapses refer to the 
1063 
times when a subject fails to respond to the task in a timely manner (i.e., > 500 msec). The test 
1064 
requires subjects to sustain attention and respond to a randomly appearing stimulus on a computer 
1065 
screen by pressing a button.  The PVT takes 10 minutes to complete and will be administered via 
1066 
computer software.  This test was developed at USARIEM and does not require any permission for 
1067 
use. 
1068 
 
1069 
Matching to Sample: The Matching to Sample Test assesses short-term spatial memory (working 
1070 
memory) and pattern recognition skills [90, 91]. The participant responds by pressing the down 
1071 
arrow key when the word "READY" appears on the screen. The participant is then presented with 
1072 
an 8 X 8 matrix of a red and green checkerboard on a color screen. The matrix is on the screen for 
1073 
4 seconds. Afterwards, the sample is removed and followed by a variable delay interval during 
1074 
which the screen is blank (except for the word delay at the bottom of the screen). After the delay, 
1075 
two matrices are presented on the screen: the original sample matrix and a second matrix that 
1076 
differs slightly in that the color sequence of two of the squares will be reversed.  The participant 
1077 
selects the comparison matrix by responding on the left or right arrow key that matches the original 
1078 
sample matrix.  A response (left or right arrow key) must be made within 15 seconds; otherwise a 
1079 
time-out error will be recorded.  Correct responses will also be recorded, as will response time to 
1080 
choose a matrix. The task lasts approximately 5 minutes and will be administered via computer 
1081 
software. After the delay, two matrices are presented: the sample matrix and a second matrix that 
1082 
differs slightly in that the color sequence of two of the squares is reversed.  The participant selects 
1083 
the comparison matrix by responding on the left or right arrow key that matches the original sample 
1084 
matrix.  A response must be made within 15 seconds; otherwise a time-out error will be recorded.  
1085 
Correct responses will also be recorded, as will response time to choose a matrix. The task lasts 
1086 
approximately 5 minutes and will be administered via computer.  This test was developed at 
1087 
USARIEM and does not require any permission for use. 
1088 
 
1089 
Grammatical Reasoning: The Grammatical Reasoning Test assesses language-based logical 
1090 
reasoning and has been used to assess the effects of various treatments on cognitive function [96].  
1091 
It has been adapted from the Baddeley Grammatical Reasoning Test.  On each trial, the letters AB 
1092 
or BA follows a statement.  The participant must decide whether or not each statement correctly 
1093 
describes the order of the two letters.  The "T" key on the keyboard is pressed for correct 
1094 
(statement is true) and the "F" key is pressed for incorrect (statement is false).  Statements can be 
1095 
positive/negative or active/passive, and a given letter may precede/follow the other letter.  A 
1096 
session lasts for 32 trials and is made up of the above combination of statements.  The time to 
1097 
complete this test is approximately 5 minutes and it will be administered via computer software.  
1098 
This test was developed at USARIEM and does not require any permission for use. 
1099 
 
1100 
The N-back Task: The N-back Task measures working memory [97]. It requires on-line monitoring, 
1101 
updating, and manipulation of remembered information and allows for the parametric assessment of 
1102 
different working memory loads. Participants will be shown a series of letters one at a time in the 
1103 
center of a computer screen and will be required to mentally take note of those depicted letters.  
1104 
They will then respond by pressing the spacebar if the letter presented is the same as the previous 
1105 
letter (1-back condition), 2 previous letters back (2-back condition), or 3 previous letters back (3-
1106 
back condition).  Dependent measures include response time and accuracy. This task takes 
1107 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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v21OCT14 
 
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approximately 15 min to complete and will be administered via computer software. This version of 
1108 
the task was developed at USARIEM and does not require any permission for use. 
1109 
 
1110 
The Balloon Analogue Risk Task: The Balloon Analogue Risk Task (BART) is a computerized 
1111 
test designed to measure willingness to take risks versus “play it safe” and requires the 
1112 
participant to fill a simulated balloon with air [98].  Points are given for keeping the balloon as full 
1113 
as possible. The more expanded the balloon gets, the more points are earned. However, all 
1114 
points are lost if the balloon is over-inflated and pops. The object of this task is to earn as many 
1115 
points as possible by keeping the balloon inflated without popping. Additionally, there is a risk-
1116 
learning component to this task as some balloon colors pop with less inflation and others with 
1117 
more, while a third category is unpredictable. Standard administration of this task allows 30 
1118 
trials.  The BART takes approximately 10 min to complete and will be administered via computer 
1119 
software.  This test is publicly available and does not require permission for use.  
1120 
 
1121 
Ambulatory Vigilance and Sleep Evaluation: The USARIEM developed wrist-worn Vigilance 
1122 
Monitoring System (produced in collaboration with PCD, Inc., Ft. Walton Beach, FL) will be used to 
1123 
continuously measure a variety of key behavioral and environmental factors while volunteers are 
1124 
engaged in daily activities.  The monitors are lightweight devices slightly larger than a wristwatch 
1125 
and are worn on the non-dominant wrist.  Each monitor contains a microprocessor, non-volatile 
1126 
memory, and several other sensors.  The monitors measure ambient temperature, sound intensity, 
1127 
and environmental light levels as well as vigilance, rest, and activity.  The monitors will be 
1128 
programmed such that at random intervals, averaging approximately 10 times an hour, an audible 
1129 
tone sequence, a light stimulus and/or a vibratory stimulus, similar to the vibration of a pager or cell 
1130 
phone, will be emitted. Vigilance during Baseline, SUSOPS and recovery will be assessed during 
1131 
specific standardized times.  The volunteer will be required to push a small button on the monitor in 
1132 
response to the stimuli.  Vigilance and response time will be assessed by monitoring correct 
1133 
responses and the latency to respond to the tone.  These measures provide estimates of different 
1134 
aspects of vigilance, the ability to detect stimuli and the ability to respond as rapidly as possible. 
1135 
Measurements of sleep and activity will be collected using the Fatigue Science ReadiBand™ 
1136 
actigraph, Philips Respironics Actiwatch® Spectrum Plus, or equivalent device. Volunteers will wear 
1137 
the monitors during the baseline and simulated SUSOPS portion of the research. 
1138 
 
1139 
B5.4 Data Collection   
1140 
Determination of Fractional Iron Absorption and Circulating Biomarkers of Inflammation 
1141 
and Muscle Damage and Nutritional, Metabolic, and Androgen Status 
1142 
 
1143 
Fasted (≥ 8 h) blood draws, and post-exercise blood draws will be collected as outlined below to 
1144 
assess  fractional iron absorption and relevant biomarkers of inflammation, muscle damage, 
1145 
appetite, and nutritional and metabolic stress responses to SUSOPS (Tables 1).  A total of 42 
1146 
blood draws (≤ ~530 mL) will be collected during the entire study.   
1147 
 
1148 
Table 1. Blood sample collection per analyte1  
1149 
 
 
SUSOPS 1 
 
Recovery 1 
SUSOPS 2 
 
Recovery 2 
Days 
2 
3 
4 
5 
6 
7 
8 
9 
12 
13 
14 
15 
16 
17 
18 
19 
22 
Tracer/tracee2 
7 
x 
x 
x 
7 
x 
x 
x 
x 
x 
x 
x 
7 
x 
x 
x 
x 
Hgb/Hct 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Erythroferrone2 
7 
x 
x 
x 
7 
x 
x 
x 
x 
x 
x 
x 
7 
x 
x 
x 
x 
c-myomiRNA 
 
x 
 
x 
x 
 
 
 
 
x 
 
x 
x 
 
 
 
 
Exo miRNA 
 
x 
 
x 
x 
 
 
 
 
x 
 
x 
x 
 
 
 
 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
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Ferritin2 
7 
x 
x 
x 
7 
x 
x 
x 
x 
x 
x 
x 
7 
x 
x 
x 
x 
sTfR 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Hepcidin2 
7 
x 
x 
x 
7 
x 
x 
x 
x 
x 
x 
x 
7 
x 
x 
x 
x 
Iron2 
7 
x 
x 
x 
7 
x 
x 
x 
x 
x 
x 
x 
7 
x 
x 
x 
x 
TIBC 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
IL-62 
7 
x 
x 
x 
7 
x 
x 
x 
x 
x 
x 
x 
7 
x 
x 
x 
x 
hs-CRP 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Erythropoietin 
 
x 
 
 
x 
 
 
 
 
x 
 
 
x 
 
 
 
 
Cortisol 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
CK 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
LDH 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
Myoglobin 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
T-test 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
SHBG 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
LH 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
DHEA-S 
 
x 
 
x 
 
x 
 
x 
x 
x 
 
x 
 
x 
 
x 
x 
Insulin3 
 
2 
 
2 
 
x 
 
x 
x 
2 
 
2 
 
x 
 
x 
x 
FFA3 
 
2 
 
2 
 
x 
 
x 
x 
2 
 
2 
 
x 
 
x 
x 
Glycerol3 
 
2 
 
2 
 
x 
 
x 
x 
2 
 
2 
 
x 
 
x 
x 
Glucose3 
 
2 
 
2 
 
x 
 
x 
x 
2 
 
2 
 
x 
 
x 
x 
β-HB3 
 
2 
 
2 
 
x 
 
x 
x 
2 
 
2 
 
x 
 
x 
x 
Osmolality 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x 
x 
Leptin3 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
 
x 
 
Acyl ghrelin3 
 
2 
 
2 
 
x 
 
x 
 
2 
 
2 
 
x 
 
x 
 
PYY3 
 
2 
 
2 
 
x 
 
x 
 
2 
 
2 
 
x 
 
x 
 
GLP-13 
 
2 
 
2 
 
x 
 
x 
 
2 
 
2 
 
x 
 
x 
 
PP3 
 
2 
 
2 
 
x 
 
x 
 
2 
 
2 
 
x 
 
x 
 
I-FABP3 
 
2 
 
2 
 
 
 
 
 
2 
 
2 
 
 
 
 
 
Zonulin3 
 
2 
 
2 
 
 
 
x 
 
2 
 
2 
 
 
 
x 
 
LBP3 
 
2 
 
2 
 
 
 
x 
 
2 
 
2 
 
 
 
x 
 
S100B3 
 
2 
 
2 
 
 
 
x 
 
2 
 
2 
 
 
 
x 
 
Ex vivo PBMC 
x 
 
 
 
x 
 
 
 
 
 
 
 
x 
 
 
 
 
Serum archive4 
8 
2 
x 
2 
8 
x 
x 
x 
x 
2 
x 
2 
8 
x 
 
x 
x 
EDTA archive4 
x 
2 
x 
2 
x 
x 
x 
x 
x 
2 
x 
2 
x 
x 
 
x 
x 
LiHep archive4 
x 
2 
x 
2 
x 
x 
x 
x 
x 
2 
x 
2 
x 
x 
 
x 
x 
mRNA-seq 
x 
 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
x 
 
Metabolomics 
x 
 
 
 
x 
 
 
x 
 
 
 
 
x 
 
 
x 
 
1Analyte abbreviations: Hbg/Hct, hemoglobin/hematocrit; c-myoMIR, circulating muscle-specific 
1150 
microRNA; sTfR, soluble transferrin receptor; T-saturation, transferrin saturation; TIBC, total iron binding 
1151 
capacity; IL-6, interleukin-6; hs-CRP, high-sensitivity C-reactive protein; CK, creatine kinase; LDH, lactate 
1152 
dehydrogenase; T-test; total testosterone; SHBG, sex-hormone binding globulin; LH, luteinizing 
1153 
hormo33.5.ne; DHEA-S, dehydroepiandrosterone-sulfate; FFA, free-fatty acids; β-HB, beta-
1154 
hydroxybutyrate; GLP-1, glucagon-like peptide-1; PYY, peptide-YY: PP, pancreatic polypeptide;  I-FABP, 
1155 
intestinal fatty acid binding protein; LBP, lipopolysaccharide binding protein, PBMC, peripheral blood 
1156 
mononuclear cells. 2Blood will be sampled seven times during the fractional iron absorption studies 
1157 
through an indwelling venous catheter for the notated analytes (0, 20, 40, 60, 120, 240, and 360 min) for 
1158 
a total of 21 blood draws (seven per iron study). A final blood draw will be taken on each of the iron study 
1159 
mornings (1 per iron study morning; 3 total) through the indwelling venous catheter that is separate from 
1160 
the seven draws for the iron studies in order to analyze the remaining markers (marked with an x on days 
1161 
2, 6, and 16) at the same time of day as those measured on the other study mornings when blood draws 
1162 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 35 of 59 
 
occur after an overnight fast (venipuncture). 3A second blood sample will be collected post-exercise for 
1163 
the notated analytes on days 3, 5, 13, and 15. 4Serum, EDTA, LiHep archived blood samples are derived 
1164 
from the blood draws at each specific time point, and stored in the event certain assays require re-
1165 
analysis, or for future analysis if any sample remains. Based on this explanation, the total number of 
1166 
study blood draws is 42 (iron study days, 8 x 3 = 24; 2 on days 3, 5, 13, and 15, for 8 total draws; and 1 
1167 
on every other indicated blood draw day, 10 total). Serum and plasma will be archived from each blood 
1168 
draw. The total volume of blood collected per participant is ≤ ~530 mL.  
1169 
 
1170 
Saliva analysis 
1171 
Saliva collection is a non-invasive technique with easy achievability that may facilitate sample 
1172 
collection in austere environments by study volunteers with minimal instruction.  The technique 
1173 
may therefore provide an efficient alternative to using blood and/or fecal collection to monitor 
1174 
physiological status in Warfighters engaged in SUSOPS or during deployment.  However, the 
1175 
validity of saliva collection as a deployable solution for monitoring physiologic status requires 
1176 
validation.  As such, saliva and blood samples will be collected in tandem.  Blood will be 
1177 
subjected to mRNA-seq, targeted micro-RNA (see mRNA, myomiR, and c-myomiR Expression 
1178 
below) and metabolomics analyses.  Saliva will be subjected to microRNA profiling, ion 
1179 
abundance measurement and metabolomics analyses.   Results will be compared between 
1180 
sample types, and in relation to the gut microbiome, and cognitive and physical performance 
1181 
outcomes.  In addition to validating the approach, analyses will be undertaken to explore 
1182 
associations between the salivary microbiome and stress responses.   
1183 
 
1184 
Saliva samples will be collected on study days 2, 6, 9, 16 and 19 at the same time blood 
1185 
samples will be collected for mRNA-seq, microRNA-seq and metabolomics assays, 
1186 
respectively.  During saliva collection, a Salivette tube with opaque white cap (SARSTEDT, Inc.) 
1187 
will be opened; the swab will be removed and placed in the mouth. After 45 seconds (gentle 
1188 
chewing possible, but not required), the saliva-soaked swab will be returned to the Salivette® 
1189 
which will be closed with the plug for storage and transport.  The expected yield is 0.8-1.4 mL 
1190 
saliva. A second round of sample collection will occur 30 minutes later following the same 
1191 
procedure. The tubes will be frozen at -80oC and shipped to USACEHR. Upon receiving the 
1192 
tubes, the tubes will be centrifuged at room temperature at 1000g for 2 minutes. The first tube 
1193 
will be used for microRNA sequencing and microbiome shot gun sequencing. The second tube 
1194 
will be used for metabolomics and targeted proteomics (inflammatory panel of 65 protein 
1195 
markers will be tested using the Luminex platform). 
1196 
 
1197 
Determination of Fractional Iron Absorption 
1198 
Stable isotope concentrations in whole blood will be determined by inductively coupled plasma 
1199 
mass spectrometry (ICP-MS) by the Energy and Environmental Sustainability Laboratory 
1200 
(EESL) at Pennsylvania State University (PSU) using a fee-for-service contract.  
1201 
 
1202 
The amount of absorbed iron in circulation will be calculated using isotope dilution as described 
1203 
previously [70, 99]. Briefly, the amount of absorbed iron circulating in blood will be calculated 
1204 
based on the amount of stable isotope administered, the amount of stable isotope detected in 
1205 
the blood, hemoglobin concentration, and blood volume, which will be estimated based on 
1206 
volunteer height and weight [100]. Isotope dilution will also be used to calculate fractional iron 
1207 
incorporation into red blood cells after administration of the stable isotope and assuming 80% 
1208 
incorporation [70].  
1209 
 
1210 
An example calculation using 54Fe tracer and endogenous concentrations of 56Fe is included 
1211 
below: 
1212 
 
1213 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 36 of 59 
 
 
1214 
54Feinc = quantity of 54Fe incorporated into red blood cells 
1215 
54Fe/56Feenr = enriched isotope ratio 
1216 
54Fe/56Febase = baseline isotope ratio 
1217 
Fecirc = total circulating iron 
1218 
NA54 = natural abundance of 54Fe 
1219 
 
1220 
Muscle Glycogen 
1221 
Approximately 20 mg of muscle from each muscle biopsy will be dehydrated in a freeze dryer.  
1222 
Samples will then be ground to powder and visible connective tissue will be removed.  
1223 
Powdered muscle will be placed in 500 µl 2 N HCl.  Samples will then be placed in an incubator 
1224 
for 120 min at 100˚C.  Following incubation samples will be neutralized with 1500 µl 0.67 N 
1225 
NaOH.  Muscle glycogen will be in solution at this point.  Glycogen will be quantified by a 
1226 
fluorometric assay (Sigma-Aldrich, St. Louis, MO, USA or equivalent).  Muscle glycogen will be 
1227 
quantified to determine the contribution of endogenous carbohydrate availability on inflammation 
1228 
in response to SUSUPS BAL and NEG BAL.       
1229 
 
1230 
mRNA, myomiR, and c-myomiR Expression 
1231 
Total RNA will be isolated from approximately 25 mg of muscle using a mirVanaTM miRNA 
1232 
isolation kit (Invitrogen, Carlsbad, CA, USA) or equivalent.  Quantity and quality of RNA will be 
1233 
assessed using a Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA).  
1234 
Equal amounts of total RNA will be synthesized into cDNA for analysis of mRNA (iScriptTM 
1235 
Advanced cDNA Synthesis Kit; Bio-Rad or equivalent) and a TaqMan® microRNA RT kit 
1236 
(Applied Biosystems, Foster City, CA, USA) or equivalent.  Individual primers will be used to 
1237 
determine the mRNA expression of known muscle inflammatory, anabolic, proteolytic, 
1238 
myogenic, and metabolic markers susceptible to stress, to include but not limited to, TNF-α, 
1239 
TNF-αR, IL-6, IL-6R, TWEAK, TWEAK-R, REDD1, Atrogin, MuRF-1, MyoD, Myogenin, 
1240 
Myogenenin, Pax7, PGC-1α, SIRT1, p53, ACC, and AMPK. microRNA analysis will be 
1241 
conducted using individual Taqman® probes (Applied Biosystems) or equivalent, assessing 
1242 
microRNA that may be associated with inflammation and metabolism.  This microRNA targets 
1243 
will include, but not be limited to, miR-1, miR-23a/b, miR-26, miR-29, miR-34a, miR-103, miR-
1244 
107, miR-133a/b, miR-146, miR-206, miR-208a, miR-486, and miR-499a. 
1245 
   
1246 
Following identification of skeletal muscle microRNA that had a significant change, Taqman® 
1247 
probes (Applied Biosystems) or equivalent will be used to assess the expression of these 
1248 
microRNA in serum to determine their potential use as noninvasive markers of altered 
1249 
metabolism in response to elevated inflammation during and following SUSOPS.  Circulating 
1250 
miRNA will be extracted from 200 µL serum using miRNeasy Serum/Plasma kit, which allows 
1251 
for extraction and purification of small (< 200 nucleotides) cell-free RNA (Qiagen, Valencia, CA, 
1252 
USA or equivalent). To avoid introduction of potentially contaminating material, prior to RNA 
1253 
extraction serum samples will be centrifuged for 10 min at 4°C to remove cellular debris.  
1254 
Supernatant will be removed and transferred to a new tube without disturbing the pellet.  Due to 
1255 
Fecirc · NA54
54Fe
56Febase
54Fe
56Feenr
54Fe
56Febase
–
54Feinc = 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 37 of 59 
 
the small amount of RNA in the serum, 3.5 µL of a Spike-In Control (C. elegans miR-39; Qiagen 
1256 
or equivalent) will be added to all samples prior to extraction of RNA to determine the yield of 
1257 
template recovered.  After extraction 3 μl of serum RNA will reverse transcribed using the 
1258 
TaqMan® microRNA RT kit (Applied Biosystems) or equivalent with miRNA-specific stem-loop 
1259 
RT primers pooled in 1X-Tris-EDTA (TE) buffer for a final dilution of 0.05X.  A pre-amplification 
1260 
step will be performed after reverse transcription to increase cDNA template using a primer pool 
1261 
of 20 X Taqman® Small RNA Assays (Applied Biosystems) or equivalent for miRNA of interest 
1262 
at 0.05X concentration in 1X TE buffer.  All serum miRNA will be normalized to the geometric 
1263 
mean of external (Spike-In Control C. elegans miR-39) and internal controls to allow for both 
1264 
technical and inter-individual normalization [101]. Geometric mean of controls will be used to 
1265 
correct for possible outlying values and abundance differences between the different controls 
1266 
[102].   
1267 
 
1268 
All reverse transcription for mRNA and miRNA, and pre-amplification of serum miRNA will be 
1269 
conducted in a T100TM Thermal Cycler (Bio-Rad, Hercules, CA or similar model). A 
1270 
StepOnePlusTM real-time PCR system (Applied Biosystems) or similar model will be used to 
1271 
perform all mRNA and miRNA analysis.  Fold changes will be calculated using the ∆∆cycle 
1272 
threshold (∆∆CT) method as described below in statistical analysis section. 
1273 
 
1274 
Bioinformatics Analysis 
1275 
microRNA with significant changes in their expression will be uploaded to DNA Intelligent 
1276 
Analysis (DIANA)-miRPath 3.0 (Alexander Fleming Biological Sciences Research Center 
1277 
[BSRC], Athens, Greece; http://diana.cslab.ece.ntua.gr) to determine potential molecular 
1278 
pathways that these microRNA have previously been reported to regulate. Relevant Kyoto 
1279 
Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) pathways will be 
1280 
identified using experimentally verified targets from TarBase 7.0 (Alexander Fleming BSRC).  
1281 
Based on findings from this analysis, any additional gene and protein expression of relevant 
1282 
targets will be assessed.    
1283 
 
1284 
Western Blotting 
1285 
Approximately 30 mg of muscle will be homogenized in ice-cold buffer (1:10 w/v) containing 50 
1286 
mM Tris–HCl (pH 7.5), 5 mM Na-pyrophosphate, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 10% 
1287 
glycerol (v/v), 1% Triton-X, 1 mM DTT, 1 mM benz-amidine, 1 mM PMSF, 10 μg mL−1 trypsin 
1288 
inhibitor and 2 μg mL−1 aprotinin.  Homogenate will be centrifuged for 15 min at 10,000 × g at 
1289 
4°C.  Protein concentration of supernatant (lysate) will be determined using 660 nm Protein 
1290 
Assay (ThermoFisher Scientific, Waltham, MA, USA or equivalent).  Phosphorylation status and 
1291 
total protein expression of intramuscular inflammation and markers of muscle carbohydrate 
1292 
metabolism will be determined by Western blot.  Muscle lysates will be solubilized in Laemmli 
1293 
buffer, with equal amounts of total protein (15 μg) separated by SDS-PAGE using precast 
1294 
Tris·HCl gels (Bio-Rad). Proteins will be transferred to polyvinylidene difluoride (PVDF) 
1295 
membranes and incubated with commercially available primary antibodies of intracellular 
1296 
markers involved in inflammation, anabolism, proteolysis, and substrate metabolism [e.g., IL-6, 
1297 
TNFα, NF-kB, IKKα/β, Akt, mTORC1, p70SK1, AMPKα, p38 MAPK, PGC-1α , SIRT1, CaMK, 
1298 
p53, and ACC (Cell Signaling Technology, Danvers, MA, USA)] at 4°C overnight.  Labeling will 
1299 
be performed using secondary antibody (anti-rabbit IgG conjugate with horseradish peroxidase; 
1300 
Cell Signaling Technology), and chemiluminescent reagent will be applied (Super Signal, West 
1301 
Pico Kit; Pierce Biotechnology, Rockford, IL, USA or equivalent). Blots will be quantified using 
1302 
the ChemiDoc XRS from Bio-Rad and Image Lab software (Bio-Rad) or similar model. To 
1303 
confirm equal protein loading per well a normalizing protein such as HSP90 or GAPDH will be 
1304 
assessed.  
1305 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 38 of 59 
 
 
1306 
B5.5 Managing Data and/or Human Biological Specimens for this Research  
1307 
 
1308 
All data and medical information obtained will be considered privileged and held in confidence.  
1309 
Study volunteers will be assigned unique subject identification (ID) numbers that will not contain 
1310 
any personal identifiers such as name, social security number, address, date of birth, zip code, 
1311 
etc.  Subject ID numbers will be used on all data collection instruments, to include 
1312 
questionnaires, data collection forms, computer records, etc.  A number will be assigned as 
1313 
each volunteer is enrolled for participation.  A master list linking the volunteers’ names and ID 
1314 
numbers will be kept in a separate locked file in the principal investigator's or the project 
1315 
coordinator’s office, or kept in a computer file with password-protected access restricted to the 
1316 
principal investigator and the project coordinator. Social security numbers and banking 
1317 
information will be collected to process volunteer payments.  The master list link, social security 
1318 
numbers, and banking information will be deleted immediately after the study has been 
1319 
completed and payment has been confirmed.  When the results of the research are published or 
1320 
discussed in conferences, no information will be included that would reveal identity.  Study 
1321 
biological samples will be processed on site at USARIEM and stored in Military Nutrition 
1322 
laboratory freezers (rooms 322, 304) using the subject identification number until analyzed on 
1323 
site or until sample aliquots are shipped to other laboratories for analysis.  Aliquots will be made 
1324 
of all study samples and stored on site in Military Nutrition laboratory freezers indefinitely for 
1325 
reanalysis if necessary.  Aliquots of coded biological samples will be shipped overnight on dry 
1326 
ice to Dr. Hammamieh at USACEHER, Dr. Rood at PBRC, Metabolic Solutions Inc., and to the 
1327 
Energy and Environmental Sustainability Laboratory (EESL PSU) at Pennsylvania State 
1328 
University for analyses.  Once samples have been analyzed by these respective laboratories, 
1329 
there will be no remaining sample for storage.  Coded data will be transmitted between the 
1330 
abovementioned laboratories and USARIEM, as well as between USARIEM and the University 
1331 
of Leeds via encrypted email, a secure file transfer site, or using an approved removable media. 
1332 
Coded specimen/data transfer agreements have been obtained for PRB and University of 
1333 
Leeds.  Further, USARIEM has existing collaborative agreements in place with the Norwegian 
1334 
Defence Research Establishment (CRADA W81XWH-12-0270) and the University of Leeds 
1335 
(W81XWH-16-0498).  An institutional collaborative agreement is currently in review between 
1336 
USARIEM and USACEHR.    
1337 
    
1338 
Only personnel assigned to the research study by the principal investigator will have access to 
1339 
the data. Only the principal investigator and project coordinator will have access to personal 
1340 
identifiable data.  No outside laboratory will have access to identifiable data.  Hard copy data 
1341 
records will be stored for a minimum of three years from the time the study is completed.  
1342 
Electronic data records will be maintained for a period of at least ten years after the study has 
1343 
been completed.   
1344 
 
1345 
B5.6 Managing Data and/or Human Biological Specimens for Future Research   
1346 
 
1347 
Any use of the samples outside of the broad scope of this protocol will be submitted as a 
1348 
protocol amendment or a new protocol.  Once the protocol is closed, samples will be retained 
1349 
for further analyses that may or may not align with the hypotheses set forth in the protocol.  
1350 
Samples will be retained for future analyses once the protocol is closed.    
1351 
 
1352 
B5.7 Devices, Drugs, Dietary Supplements, Nutritional Supplements, And Biologics   
1353 
 
1354 
B5.7.1 Devices 
1355 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 39 of 59 
 
 
1356 
5.7.1.1 FDA-approved device being used in this research according to the 
1357 
approved labeling  
1358 
 
1359 
SmartPill, Polar Electro heartrate monitor, True Max 2400 ParvoMedics, Fatigue 
1360 
Science ReadiBandTM actigraph, Philips Respironics Actiwatch® Spectrum Plus, 
1361 
InBody 720, and DEXA will be used in research according to approved labeling. 
1362 
 
1363 
5.7.1.2 FDA-approved device being used in this research in a manner other 
1364 
than its approved labeling 
1365 
N/A 
1366 
B5.7.2 Drugs 
1367 
                              
1368 
B5.7.2.1 FDA-approved and used in accordance with the approved labeling 
1369 
N/A 
1370 
 
1371 
B5.7.2.2 FDA-approved and used in a manner not in accordance with its 
1372 
approved labeling 
1373 
N/A 
1374 
 
1375 
B5.7.2.3 Any drug not approved by the FDA 
1376 
N/A 
1377 
 
1378 
B5.8 Statistical Analysis 
1379 
 
 
1380 
B5.8.1 Sample Size Estimation 
1381 
 
1382 
Power analyses were performed using www.biomath.info/power/prt.htm and indicate that 
1383 
11 participants will provide adequate power (alpha = 0.05, power = 0.8) to detect 
1384 
significant changes in circulating concentrations of IL-6 and hepcidin in response to 
1385 
stress and differences in energy balance. To account for potential attrition (~30% attrition 
1386 
in 16-02-HC), 4 additional subjects are requested for a total of 15 subjects.  Data used in 
1387 
calculating samples size estimates (i.e., means and standard deviations) are derived 
1388 
from previous studies that found significant increases in IL-6 and/or hepcidin in 1) male 
1389 
athletes (n=12) after a 7-day training block [103], 2) male athletes (n=9) after two 75 min 
1390 
endurance exercise bouts separated by 3 h of rest [22], 3) male athletes (n=8) after two 
1391 
high-intensity endurance exercise bouts over two days [23], and 4) male athletes (n=11) 
1392 
completing a 1.5 h treadmill run [104]. The outcome measures, means, standard 
1393 
deviations (SD), n, and alpha and power levels are included below: 
1394 
 
1395 
Study 
Outcome 
Mean 
difference 
SD of 
difference 
Alpha 
Power 
n/group 
[103] 
Hepcidin 
4.7 
4.9 
0.05 
0.8 
11 
 
IL-6 
2.7 
1.3 
0.05 
0.8 
5 
[22] 
IL-6 
4.8 
1.8 
0.05 
0.8 
6 
[23] 
IL-6 
3.2 
2.0 
0.05 
0.8 
6 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 40 of 59 
 
[104] 
Hepcidin 
7 
7.4 
0.05 
0.8 
11 
 
IL-6 
9 
6.6 
0.05 
0.8 
7 
 
1396 
We have previously shown that an increase of intestinal permeability of 55-60% is 
1397 
associated with inflammation in military-relevant environments (USARIEM protocol 14-
1398 
33HC [44]).  Using means (pre-SUSOPS sucralose excretion = 2.0%), standard 
1399 
deviations (SD = 1.1), and correlations for repeated measurements of sucralose 
1400 
excretion (r = 0.5) from 14-33HC, we estimate that 14 subjects will allow detection of a 
1401 
main effect of study phase (BL vs. SUSOPS-NEG BAL vs. SUSOPS-BAL) if the effect 
1402 
size of SUSOPS-BAL vs. SUSOPS-NEG BAL on intestinal permeability is medium 
1403 
(~20% decrease)  at power = 0.80 and alpha = 0.017 
1404 
(https://glimmpse.samplesizeshop.org/).  Of note, larger effect sizes have been reported 
1405 
in studies investigating the effects of nutritional interventions on intestinal permeability 
1406 
during exercise [105].  Additionally, we feel that it is likely that the SDs measured in this 
1407 
laboratory study will be slightly lower than those measured in our previous study (14-
1408 
33HC) which was conducted in a field environment.  A 25% decrease in the SD would 
1409 
allow us to detect a main effect of phase with 11 subjects at power = 0.80 and alpha = 
1410 
0.017.  As such we expect an n of 11 to 15 will provide adequate power to detect a main 
1411 
effect of phase on intestinal permeability.   
1412 
 
1413 
Power calculations for appetite-mediating hormones outcomes were conducted using 
1414 
data obtained during USARIEM protocol H10-09 [73, 106].  In that study an energy 
1415 
deficit (3700 kcal/d) slightly greater than that which will be imposed in this study resulted 
1416 
in a 2.5-fold decrease in fasted acyl ghrelin, a 2.2 fold decrease in leptin, a 1.7-fold 
1417 
decrease in PYY, and a 3-fold increase in PP concentrations concomitant to an ~11-fold 
1418 
increase in appetite and overeating during recovery from energy deficit.  Based on these 
1419 
results we expect to observe the hormone responses in the table below where fasting 
1420 
concentrations will be approximately maintained during BAL, and the maximal difference 
1421 
between BAL and NEG BAL will be observed on SUSOPS day 3 followed by a full 
1422 
recovery of hormone concentrations by recovery day 3.  For all but PP, 12 subjects will 
1423 
provide ≥80% power at alpha = 0.01 to detect a phase-by-time interaction. 
1424 
 
BAL 
NEG BAL 
 
 
 
 
S-d1 
S-d3 
R-d1 
R-d3 
S-d1 
S-d3 
R-d1 
R-d3 
SD 
r 
n 
Ghrelin 
185 
185 
185 
185 
185 
75 
135 
185 
90 
P,0.65; 
T, 0.60 
11 
Leptin 
9 
9 
9 
9 
9 
4.1 
6.5 
9 
5 
P,0.60; 
T, 0.75 
11 
PYY 
170 
170 
170 
170 
170 
100 
135 
170 
170 
P,0.85; 
T, 0.85 
12 
PP 
65 
65 
65 
65 
65 
195 
130 
65 
100 
P,0.30; 
T, 0.30 
19 
Samples sizes (n) calculated using https://glimmpse.samplesizeshop.org/. r, correlation between phases (P) 
1425 
and over time within each phase (T); R, recovery; S, SUSOPS; SD, standard deviation. Values based on 
1426 
those measured during USARIEM protocol H10-09 [109]]. 
1427 
 
1428 
 
1429 
Using data obtained using the SmartPill in a study of 73 healthy adults we estimate that 
1430 
mean gastrointestinal transit time at baseline will be 29.5 hr (SD = 11.0 hr) [107].  Horner 
1431 
et al. [108] reported a large effect size of 1.0 in a meta-analysis of high-quality studies 
1432 
that measured changes in orocecal transit time in response to exercise.  A similar effect 
1433 
size was reported by Corvilain et al. [109] who measured gastric emptying in healthy 
1434 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 41 of 59 
 
adults before and after a 4-d fast.  Using these data and a conservative correlation 
1435 
between repeated measurements of r = 0.50 (Diaz-Tartera et al. [107] reported r = 0.99), 
1436 
we estimate that n = 8 participants will provide >80% power to detect a main effect of 
1437 
condition at alpha = 0.05.  This sample size will also provide >80% power to detect a 
1438 
main effect should the effect size be medium (effect size = 0.5) and the correlation 
1439 
between repeated measurements (r = 0.90) more similar to that reported by Diaz-Tartera 
1440 
et al. [107]. We will enroll at least n = 9 in this procedure to ensure complete data is 
1441 
collected on 8 participants.  Due to equipment availability, n = 9 is also the maximum 
1442 
number of participants we can measure simultaneously.  However, should multiple 
1443 
iterations be needed to complete data collection, we will enroll up to n = 9 participants 
1444 
during each iteration.  Although n = 8 is sufficient to detect a main effect of condition, 
1445 
enrolling more than n = 9 will provide greater power to detect associations between 
1446 
changes in gastrointestinal transit time and changes in gut microbiota composition, 
1447 
intestinal permeability, and appetite-related outcomes.  
1448 
 
1449 
Next Generation Sequencing (maximum 200 million pair-end reads) of blood mRNA 
1450 
samples from 15 participants will yield 60% statistical power to detect a true difference in 
1451 
expression of at least 1.5 fold with group coefficient variation 0.4, and  a conservative 
1452 
false discovery rate of 0.05 using moderated t-tests and with transcriptome coverage of 
1453 
9 over reference human genome with sequence length of 150 base pairs. Note, a more 
1454 
liberal false discovery rate (e.g., 0.20) may be used to reduce type 2 error rates and 
1455 
increase power.  For microbiome analysis, shotgun sequencing (maximum 50 million 
1456 
pair end reads) of 15 participants will achieve ≥69% power to detect a true difference in 
1457 
relative abundance of at least 1.5 fold with group coefficient variation 1.3, and false 
1458 
discovery rate of 0.05. Again, a more liberal false discovery rate (e.g., 0.20) may be 
1459 
used to reduce type 2 error rates and increase power.  Power calculations were 
1460 
conducted using pairwise distances and PERMANOVA power with respect to effect size. 
1461 
However, power will be enhanced by correlating the blood mRNA data with other omics 
1462 
readouts derived from saliva (microRNA-seq, metabolomics and microbiome) and blood 
1463 
(targeted microRNA and metabolomics), and by temporal sample collection.  As such, 
1464 
we expect that 15 participants will provide adequate power to detect differences in blood 
1465 
mRNA expression, and gut microbiome community and genome composition between 
1466 
study phases, and correlations between these parameters and other –omics readouts. 
1467 
   
1468 
B5.8.2 Data analysis  
1469 
 
1470 
Statistical analyses will be conducted using either SPSS (IBM Corp. Armonk, NY), SAS 
1471 
9.3 (SAS Institute Inc., Carey, NC), or equivalent.  Common descriptive statistics will be 
1472 
used to describe volunteer characteristics.  Shapiro-Wilk tests will be used to determine 
1473 
normality of data, and transformations will be applied as appropriate to ensure model 
1474 
assumptions are met.  Correlation and multiple regression will be used to evaluate 
1475 
relationships between study outcomes.   
1476 
 
1477 
Primary Objectives 
1478 
Linear mixed models will be used to determine main effects of phase (SUSOPS BAL and 
1479 
SUSOPS NEG BAL), time within phase, and their interaction on inflammation, iron 
1480 
absorption, mood/cognitive, and measures of muscle, nutritional, and metabolic 
1481 
homeostasis.  If phase by time interactions are observed, a Bonferroni correction will be 
1482 
applied for multiple comparisons.  Statistical significance will be set at P < 0.05. To test 
1483 
for carryover effects a main effect of group (SUSOPS BAL first, SUSOPS NEG BAL first) 
1484 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 42 of 59 
 
and a group-by-phase interaction will be included in initial models.  These terms will be 
1485 
removed if they do not significantly contribute to the overall fit of the model.   When 
1486 
significant phase-by-time interactions are observed post hoc comparisons will be made 
1487 
using paired t-tests with Bonferroni corrections.   
1488 
 
1489 
Secondary Objectives 
1490 
Linear mixed models will be used to examine changes in intestinal permeability and 
1491 
gastrointestinal transit time. Models will include subject as a random factor, and study 
1492 
phase as a fixed factor.  To test for carryover effects a main effect of group (SUSOPS 
1493 
BAL first, SUSOPS NEG BAL first) and a group-by-phase interaction will be included in 
1494 
initial models.  These terms will be removed if they do not significantly contribute to the 
1495 
overall fit of the model.  When a significant main effect is observed post hoc 
1496 
comparisons will be made using paired t-tests with Bonferroni corrections.  
1497 
 
1498 
Fecal metabolomics data will be visualized using hierarchical average-linkage clustering 
1499 
and principal components analysis. Bacterial taxonomic data will be visualized using 
1500 
hierarchical average-linkage clustering and principal coordinates analysis of beta (i.e., 
1501 
between samples) diversity scores (e.g., Bray-Curtis, and weighted and unweighted 
1502 
UniFrac). Alpha (i.e., within-sample) diversity will be calculated for taxonomic data using 
1503 
Shannon, Simpson and Chao1 indices.  Linear mixed models will be used to examine 
1504 
changes in biomarkers of gut health, fecal metabolite concentrations, and alpha diversity 
1505 
over time. Models will include subject as a random factor, and study phase, time within 
1506 
phase, and their interaction as fixed factors.  To test for carryover effects a main effect of 
1507 
group (SUSOPS BAL first, SUSOPS NEG BAL first) and a group-by-phase interaction 
1508 
will be included in initial models.  These terms will be removed if they do not significantly 
1509 
contribute to the overall fit of the model.  Models for bacterial taxa and gene abundance 
1510 
will be analyzed using the R statistical software package “DESeq2” or equivalent.  The 
1511 
Benjamini-Hochberg correction will be used to control the false discovery rate in 
1512 
microbiome-specific and metabolite models.  Data analysis will be completed using 
1513 
SPSS, XLSTAT, R, Qiime, or similar software as needed. 
1514 
 
1515 
Linear mixed models will be used to examine changes in appetite-mediating hormone 
1516 
concentrations, appetite ratings, food preferences, food choice during recovery (i.e., diet 
1517 
macronutrient proportion and energy intake). Models will include subject as a random 
1518 
factor, and study phase, time within phase, and their interaction as fixed factors.  
1519 
Separate analysis will be conducted for fasted and post-exercise appetite-mediating 
1520 
hormones.  To test for carryover effects a main effect of group (SUSOPS BAL first, 
1521 
SUSOPS NEG BAL first) and a group-by-phase interaction will be included in initial 
1522 
models.  These terms will be removed if they do not significantly contribute to the overall 
1523 
fit of the model.   When significant phase-by-time interactions are observed post hoc 
1524 
comparisons will be made using paired t-tests with Bonferroni corrections.  
1525 
 
1526 
The mRNA seq strand-specific libraries will be assayed using the Illumina HiSeq 
1527 
platform. Paired end reads of the strand-specific RNA sequences (RNA-Seq) will be 
1528 
generated using NextSQ from one flow cell with eight lanes, producing at least 30 million 
1529 
reads of 100 bases per sample. Based calling and expression analysis will be conducted 
1530 
using the Illumina-provided tool CASAVA. miRNA (20-40 bp) will be size selected from 
1531 
total RNA and will be processed by Illumina NextSQ. Image analysis and base calling 
1532 
will be performed using the newest available version of the Illumina pipeline. 
1533 
Preprocessing of raw base calls and sample de-multiplexing will be performed using the 
1534 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 43 of 59 
 
standard open source tool CASAVA. The microRNA read count matrix will be generated 
1535 
by a series of tool kits of latest version including CutAdapt, short read aligner Bowtie and 
1536 
PICARD. Metabolomics and proteomics reads will be processed by the software offered 
1537 
by the vendors namely Waters Corporation and BioRad, respectively. 
1538 
 
1539 
Using a systems approach, we will integrate multiple levels of biological information. We 
1540 
have published the analysis pipeline and implemented it successfully in the past. Our 
1541 
pipeline uses industry standards and our established SOPs. We will start by analyzing 
1542 
and processing data at the analyte level for each data type such as mRNA expression, 
1543 
miRNA expression, microbiome, proteomics, and metabolomics. We will first examine 
1544 
the confounding factors or batch effects caused by non-biological factors, such as the 
1545 
technical error, sample processing date etc., using principal component analysis and 
1546 
ANOVA. If necessary, corrections will be made using the COMBAT algorithm. All 
1547 
datasets will be visually inspected using PCA, heatmaps, and boxplots before and after 
1548 
any required normalization, correction, or filtering, to assure quality control. 
1549 
 
1550 
Using R package the significantly altered analytes will be mined by t-test p-values 
1551 
(<0.05) and fold-change 1.5. The R package BETR in conjunction with standard and 
1552 
lagged correlation analysis will be used to find biological signatures which vary 
1553 
systematically with the phenotypic data. Data dimension reduction, specifically latent 
1554 
factor analysis for phenotypic (clinical indices of disease onset) and PCA/PCoA for 
1555 
omics data will be used to uncover underlying mechanisms and dominating trends. Venn 
1556 
diagrams will be used to identify the biological signatures, including the molecules and 
1557 
enriched networks/pathways, which will emerge common/unique to different treatment 
1558 
groups and common/unique to control vs. treated specimens. To assess gene ontology 
1559 
and pathway enrichments, and analyze regulatory networks and common pathways we 
1560 
will use the Hypergeometric Test (false discovery rate, q < 0.05) of Bingo 2.44 and 
1561 
ClueGO (Cytoscape 3.0.1 plugins; http://www.cytoscape.org/), Fisher’s Exact Test of 
1562 
Ingenuity Pathway Analysis (IPA, Ingenuity, Inc., Redwood, CA), and Gene Set 
1563 
Enrichment Analysis (http://www.broadinstitute.org/gsea/index.jsp) software sets. Gene-
1564 
metabolite condition ‘interactomes’ will be constructed and visualized using Gephi.0.8.2 
1565 
beta (www.gephi.org). Using a suite of platforms such as ClueGo, Bingo, IPA, David and 
1566 
IMPaLA, we will deliver a panel of networks informed by multiple levels of molecular 
1567 
evidence that are unique to treatment groups. From these analyses we will produce a 
1568 
suite of networks informed by multiple levels of molecular evidence that are unique to 
1569 
treatment groups. 
1570 
 
1571 
We will employ our in-house developed tool, Core Module Biomarker Identification with 
1572 
Network Exploration (COMBINER), to look for those novel functional groupings of 
1573 
molecules, which are statistically perturbed across time course.  In addition, Feature 
1574 
Assisted Clustering for Time-series (FACT), a pipeline for exploratory analysis and 
1575 
visualization of longitudinal data, will identify differential expressed molecules, 
1576 
pathways/GO terms, separate clusters of similar patterns, and compare pathway 
1577 
dynamics.  Finally, we will use commonly accepted criteria of Receiver Operator Curve 
1578 
(ROC), using Area Under Curve (AUC), sensitivity, and specificity to evaluate the 
1579 
performance of individual molecules.       
1580 
 
1581 
 
1582 
 
1583 
SECTION C:  HUMAN RESEARCH PROTECTIONS 
1584 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 44 of 59 
 
 
1585 
C1.  RECRUITMENT AND CONSENT   
1586 
 
1587 
C1.1 Identification and Selection of Subjects  
1588 
 
1589 
Interested volunteers who have been briefed on study procedures will be provided the 
1590 
opportunity to consent to participate.  After consent, and before medical clearance, study 
1591 
eligibility will be determined based on volunteer responses to questions pertaining to 
1592 
self-reported study inclusion and exclusion criteria (see “Background Questionnaire”). If 
1593 
still eligible, volunteers will make an appointment for a medical screening. If an individual 
1594 
fails the screening, their screening and demographic data will be destroyed. An 
1595 
additional demographic and nutritional questionnaire will be administered during the pre-
1596 
study baseline period to those participants who are enrolled in the study (see “Additional 
1597 
Demographic and Nutritional Survey”). 
1598 
  
1599 
The medical clearance will take place at USARIEM (Natick, MA) by OMSO staff.  
1600 
Volunteers recruited through SSIT may undergo OMSO medical clearance at their home 
1601 
duty station prior to arrival at USARIEM (clearances will be coordinated between the PI, 
1602 
OMSO, and the unit’s Brigade Surgeon). The clearance will include a blood draw to 
1603 
assess health status and inclusion/exclusion criteria. The medical screening visit will 
1604 
take approximately 1 hr. If any medical screening tests show a possible medical 
1605 
concern, the volunteer will be notified. Those who receive study clearance and meet the 
1606 
inclusion/exclusion criteria will continue on pre-study, baseline testing.  
1607 
 
1608 
C1.2 Recruitment Process   
1609 
 
1610 
Volunteers will be recruited from the Natick Human Research Volunteer (HRV) Pool, and 
1611 
the active duty population located at other military organizations, to include coordination 
1612 
with NSSC Soldier/Squad Interface Team (SSIT).  
1613 
 
1614 
For HRVs, the Principal Investigator will provide a copy of the informed consent 
1615 
document to the HRV Program Coordinator or designee. The Coordinator will schedule 
1616 
the consent briefing for the military human research volunteer platoon and will serve as 
1617 
ombudsman during the briefing. The HRV Coordinator may also organize consent 
1618 
briefings for Soldiers at their Advanced Individual Training unit.  The Coordinator will 
1619 
serve as an ombudsman for the offsite consent briefings.  In addition, other military 
1620 
organizations may be recruited through coordination with NSSC SSIT.  The NSSC SSIT 
1621 
Coordinator will schedule the consent briefing for the military research volunteers and an 
1622 
ombudsman will be present during briefing. Ms. Katelyn Guerriere will serve as the 
1623 
ombudsman for the current study.  
1624 
 
1625 
Superiors of Service members (e.g., unit officers, senior NCOs, and equivalent civilians) 
1626 
shall not be present at any recruitment sessions or during the consent process in which 
1627 
members of units under their command are afforded the opportunity to participate as 
1628 
human subjects of research. 
1629 
 
1630 
Other active duty personnel may also be recruited by “word of mouth” and posted flyers   
1631 
Recruiting materials will be distributed around NSSC. The text-based flyer will be posted 
1632 
on various USARIEM social media sites and used in distribution media requiring a text 
1633 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 45 of 59 
 
format (e.g., electronic newsletters).  Approvals from the requisite parties will be 
1634 
obtained prior to any recruitment activities.  
1635 
 
1636 
C1.3 Eligibility   
1637 
All potential volunteers will complete the background questionnaire pertaining to the 
1638 
study inclusion and exclusion criteria. Volunteers must then be medically cleared by 
1639 
OMSO for participation in accordance with USARIEM procedures outlined for screening 
1640 
volunteers for research involving exercise. Volunteers recruited through SSIT may 
1641 
undergo OMSO medical clearance at their home duty station prior to arrival at USARIEM 
1642 
(clearances will be coordinated between the PI, OMSO, and the units Brigade Surgeon). 
1643 
Volunteers will be screened for anemia and problems with blood clotting, including 
1644 
prothrombin time (PT)/ partial thromboplastin time (PTT), which is a specific criterion for 
1645 
research involving muscle biopsies. Health problems identified during the screening 
1646 
process will be documented and a copy provided to the volunteer. The volunteer will be 
1647 
encouraged to make an appointment with their primary care provider for a full evaluation 
1648 
of the problem. Volunteers with evidence of any physical, mental, and/or medical 
1649 
conditions that would make the proposed studies relatively more hazardous will be 
1650 
excluded. Any personal health information collected during this screening process will be 
1651 
destroyed at the time of study withdrawal or at the completion of the study. 
1652 
 
1653 
All volunteers must be willing to consume only food and beverages provided by study 
1654 
staff during the SUSOPS phases of the protocol, and they must be willing to adhere to 
1655 
exercise and physical activity prescriptions and restrictions.  If the results of all screening 
1656 
tools reveal the volunteer fits the screening criteria, they will be eligible to volunteer for 
1657 
the study.   
1658 
 
1659 
C1.4 Consent Process 
1660 
 
1661 
Prior to providing informed consent, discussions with potential volunteers (such as over 
1662 
the telephone) will not involve the collection of any personally identifiable information 
1663 
besides their name, email, and telephone number. The principal investigator, an 
1664 
associate investigator or the project coordinator will brief potential volunteers about the 
1665 
nature, purpose, procedures involved, risks, expectations and requirements for 
1666 
participation in the study. Study briefings will be scheduled to occur in-person, and will 
1667 
not occur over the phone.  Prospective volunteers will be familiarized with the study 
1668 
procedures and informed verbally and in writing of their rights to withdraw from any part 
1669 
of the study without penalty or prejudice. The principal investigator or designee will 
1670 
answer all group and private questions. Potential volunteers will have at least one hour 
1671 
after they are briefed, with the ombudsman remaining present, to read and review the 
1672 
Informed Consent document and decide whether they wish to consent to participate. No 
1673 
study procedures will occur prior to any volunteer giving their informed consent.  An 
1674 
ombudsman will not be required for any individual briefings. A copy of the informed 
1675 
consent document will be provided to the volunteer with the original kept for study 
1676 
documentation. If they meet all the medical selection and eligibility criteria after 
1677 
completing the screening health assessment and consenting to participate, they will 
1678 
begin preliminary testing. Volunteers who have already consented will be informed of 
1679 
any new information or changes to the protocol that may affect their willingness and 
1680 
ability to continue participation in the study using an approved consent addendum. 
1681 
 
1682 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
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C1.4.1 Research involving subjects with cognitive impairment or who lack 
1683 
capacity to provide informed consent 
1684 
N/A 
1685 
 
1686 
C1.4.2 Research involving non-English speaking subjects 
1687 
N/A 
1688 
 
1689 
C1.4.3 Research involving a waiver of the requirement to obtain informed 
1690 
consent OR alteration of the elements of informed consent 
1691 
N/A 
1692 
 
1693 
C1.4.4 Research involving a waiver of the requirement for investigator to 
1694 
obtain a signed consent form 
1695 
N/A 
1696 
 
1697 
C1.4.5 Waivers of assent or parental permission when the research 
1698 
 involves children 
1699 
N/A 
1700 
 
1701 
C1.4.6 Research involving data collection for the USAMRDC Volunteer 
1702 
 Registry Database 
1703 
It is the policy of USAMRDC that data sheets are to be completed on all 
1704 
volunteers participating in this research for entry into the U.S. Army Medical 
1705 
Research and Development Command Volunteer Registry Database.  The 
1706 
information to be entered into this confidential database includes name, address, 
1707 
social security number, study name, and dates.  The intent of the database is 
1708 
twofold: first, to readily answer questions concerning an individual’s participation 
1709 
in research sponsored by the USAMRDC; and second, to ensure that the 
1710 
USAMRDC can exercise its obligation to ensure research volunteers are 
1711 
adequately warned (duty to warn) of risks and to provide new information as it 
1712 
becomes available.  The information will be stored at the USAMRDC for a 
1713 
minimum of 75 years. 
1714 
 
1715 
C2. COMPENSATION FOR PARTICIPATION   
1716 
 
1717 
Volunteers will receive a total of $1050 for completing the study.  This is based on an amount of 
1718 
$25 per blood draw (42 total draws).  
1719 
 
1720 
Note: Volunteers who receive more than $600 in a calendar year will have this income reported 
1721 
to the Internal Revenue Service.   
1722 
 
1723 
C3. WITHDRAWAL FROM RESEARCH PARTICIPATION  
1724 
 
1725 
Volunteers will be allowed to withdraw at any time without penalty or loss of benefits to which 
1726 
they would otherwise be entitled. An investigator may stop an individual’s participation in the 
1727 
study if the volunteer is unwilling or unable to complete study procedures or follow study 
1728 
diets/exercise prescriptions. An investigator may also withdraw a volunteer if the individual 
1729 
becomes ill or injured or it would not be in the volunteer’s best interest to continue. If the 
1730 
participant is withdrawn by the investigator or decides to voluntarily withdraw himself, all further 
1731 
data collection will discontinue, but the data that was collected up to the point of withdrawal may 
1732 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
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still be used for analysis.  Participants will be compensated for any blood draws they completed 
1733 
up until that point, and they will be asked to return any study food and/or wrappers, study 
1734 
supplies that were provided, in addition to any study logs that they had completed up to the 
1735 
point of withdrawal.  
1736 
 
1737 
C4. PRIVACY FOR SUBJECTS   
1738 
 
1739 
To protect the volunteer’s privacy, all of their research-related records will be labeled or “coded” 
1740 
with an assigned research volunteer number that will not include their name or any other form of 
1741 
identifiable information.  The principal investigator or project coordinator will keep the link 
1742 
between volunteer number and the volunteer’s research records in a locked cabinet.  Any 
1743 
documents that will require the volunteer’s name, such as the consent form, will be kept in a 
1744 
locked cabinet separate from any research documents that contain the volunteer’s ID number.  
1745 
The principal investigator and project manager are the only people who will be able to match the 
1746 
research volunteer number with any of their personal identifying information.    
1747 
 
1748 
When the results of the research are published or discussed in conferences, no information will 
1749 
be included that would reveal the volunteer’s identity to others.  If photographs, videos, or audio-
1750 
tape recordings of volunteers are used for educational purposes, volunteer identity will be 
1751 
protected or disguised.  All identifiable or recognizable information (e.g., names and faces) will 
1752 
be covered in any photographs unless volunteers agree to sign a photo release form. If 
1753 
volunteers do not sign a photo release form, any photographs taken of them will be destroyed. 
1754 
 
1755 
C5.  CONFIDENTIALITY PROCEDURES FOR RESEARCH RECORDS, DATA, HUMAN 
1756 
BIOLOGICAL SPECIMENS  
1757 
 
1758 
All data and medical information obtained will be considered privileged and held in confidence.  
1759 
Study volunteers will be assigned unique subject identification (ID) numbers that will not contain 
1760 
any personal identifiers such as name, social security number, address, date of birth, zip code, 
1761 
etc.  This study subject ID number will be used on all data collection instruments, to include 
1762 
questionnaires, data collection forms, computer records, etc.  A number will be assigned as 
1763 
each volunteer is medically cleared for participation.  A master list linking the volunteers’ names 
1764 
and ID numbers will be kept in a separate locked file in the principal investigator's or project 
1765 
manager’s office, or kept in a computer file with password-protected access restricted to the 
1766 
principal investigator and project manager.  When the results of the research are published or 
1767 
discussed in conferences, no information will be included that would reveal identity.  Study 
1768 
samples will be processed on site at USARIEM, and off-site at PBRC, USACEHR, and at the 
1769 
fee-for-service labs.  All samples will be stored using the subject identification number.  The 
1770 
volunteers name or other identifiable information will not be included on any data, data 
1771 
collection sheets, specimens, or other research records.  Coded data from the LFPQ will be 
1772 
provided to the University of Leeds for analysis using encrypted email or a secure file transfer 
1773 
website (https://safe.amrdec.army.mil/SAFE/ Welcome.aspx).  Coded study data may be shared 
1774 
with the USACEHR for integration with –omics data sets.  Any shared data will be transferred 
1775 
using encrypted email, a secure file transfer website 
1776 
(https://safe.amrdec.army.mil/SAFE/Welcome.aspx) or the SysBioCube platform 
1777 
(https://sysbiocube-abcc.ncifcrf.gov/).  No personally identifiable information will be shared with 
1778 
PBRC, the University of Leeds, or USACEHR.      
1779 
 
1780 
Only personnel assigned to the research study by the principal investigator will have access to 
1781 
the data.  Only the principal investigator and project coordinator will have access to personal 
1782 

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identifiable data.  Hard copy data records will be stored for a minimum of three years from the 
1783 
time the study is completed.  Electronic data records will be maintained for a period of at least 
1784 
ten years after the study has been completed.   
1785 
 
1786 
C6.  RISKS OF HARM, MEASURES TO REDUCE THE RISKS OF HARM, AND BENEFITS 
1787 
OF PARTICIPATION 
1788 
 
1789 
C6.1 Risks of Harm   
1790 
 
1791 
Research Procedure Name: Venipuncture 
1792 
Research Procedure Description: A needle is used to draw blood from a superficial 
1793 
vein.   
1794 
Research-related Risks:  Venipuncture is a routine clinical procedure the medical 
1795 
community commonly uses to obtain blood samples. The risks of venipuncture are small 
1796 
and usually limited to local bruising or swelling. Sometimes volunteers feel faint or may 
1797 
faint during or right after venipuncture.  If the volunteer has had problems with fainting 
1798 
during blood draws in the past, they may be more prone to them during future 
1799 
procedures. Dizziness or faintness constitutes no long-term harm, and immediate relief 
1800 
is achieved by having the subject put their head down between their knees or lie down. 
1801 
In addition, a hematoma may result from the venipuncture, but this is more unsightly 
1802 
than risk producing.  Late complications might include thrombosis of the vein due to 
1803 
trauma or infection. These complications are extremely rare.  
1804 
Measures to Minimize Risks of Harm:  Volunteer monitoring, aseptic technique, 
1805 
including sterile disposable blood collection apparatus and adherence to standard 
1806 
medical precautions reduce risk. Trained technicians will perform all venipuncture. 
1807 
         
1808 
Research Procedure Name:  Venous Catheterization 
1809 
Research Procedure Description:  A needle will be used to guide a catheter into a 
1810 
superficial vein in the antecubital fossa (or distally). The catheter will either be attached 
1811 
to saline, or flushed periodically with saline, to keep the line patent for serial blood 
1812 
draws.     
1813 
Research-related Risks:  The risks of venous catheterization are small and usually 
1814 
limited to local bruising or swelling.  Sometimes volunteers feel faint or may faint during 
1815 
or right after the catheter is placed.  If the volunteer has had problems with fainting 
1816 
during blood draws in the past, they may be more prone to them during future 
1817 
procedures. Dizziness or faintness constitutes no long-term harm, and immediate relief 
1818 
is achieved by having the subject put their head down between their knees or lie down. If 
1819 
the catheter becomes clogged at any time during the protocol, it will be replaced to 
1820 
continue blood sampling and therefore the study. This will require another needle to be 
1821 
inserted.   
1822 
Measures to Minimize Risks of Harm: Trained technicians will use aseptic techniques 
1823 
to place the catheter; however, in spite of being careful there is a chance that the site 
1824 
may become infected. Volunteers should not give blood for 4 months before and 2 
1825 
months after the study.   
1826 
 
1827 
Research Procedure Name: Oral Stable Isotope Administration 
1828 
Research Procedure Description: Volunteers will consume a drink containing stable 
1829 
isotopically labeled iron or amino acids on multiple occasions.   
1830 
Research-related Risks: There are no known risks or reported side effects associated 
1831 
with oral administration of stable isotopes to humans during clinical or experimental 
1832 

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whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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studies.  This is because there is relatively little mass difference between the isotopic 
1833 
tracers and the more prevalent natural isotopes, and the body’s naturally occurring pool 
1834 
of stable isotopes is high enough that types of experimental protocols proposed have no 
1835 
appreciable effect on the total abundance of the isotopes present in the body.  
1836 
Measures to Minimize Risks of Harm: All staff who directly participates in the stable 
1837 
isotope studies will be properly trained to prepare and administer from Dr. Pasiakos, who 
1838 
has extensive experience with stable isotopes.  
1839 
 
1840 
Research Procedure Name:  Percutaneous Skeletal Muscle Biopsy 
1841 
Research Procedure Description: A small incision will be made in the skin and fascia 
1842 
of the vastus lateralis.  A 5-mm Bergstrom biopsy needle will pass through these 
1843 
incisions with manual suction applied to collect muscle samples, while the volunteer is 
1844 
under local anesthesia (1% lidocaine). 
1845 
Research-related Risks:  Percutaneous needle muscle biopsies have been established 
1846 
as a non-routine, but safe research procedure. Similar to blood draws, there is a risk that 
1847 
volunteers will feel faint or may faint right after a muscle biopsy.  If the volunteer has had 
1848 
problems with fainting during blood draws or muscle biopsies in the past, they may be 
1849 
more prone to them during future procedures.  The most common risks associated with 
1850 
muscle biopsies are pain (~1.27%), erythema (~1.27%), and ecchymosis (1.27%).[110, 
1851 
111] Panic episode, bleeding, and edema have also been reported (0.21%, 0.42%, and 
1852 
0.84%, respectively).[110] Denervation, numbness, and atrophy may occur but have not 
1853 
been verified in the literature.  Some minimal scarring will accompany healing of the 
1854 
incision and formation of a hypertrophic scar or keloid is possible.  Although this is a rare 
1855 
event in fair-skinned persons, the incidence of hypertrophic scarring or keloid formation 
1856 
associated with healing of a primarily closed skin biopsy site (i.e., one which was closed 
1857 
with sutures immediately afterward) is 5-10% in dark-skinned persons.   
1858 
Measures to Minimize Risks of Harm: Complications of bleeding can be reduced by 
1859 
applying direct pressure to the wound following the biopsy.  If symptoms should occur, 
1860 
they usually do not interfere with normal walking or heavier exercise.  Volunteers with 
1861 
evidence of bleeding diathesis should be excluded during medical clearance; those with 
1862 
local skin infection or irritation or recent use of anticoagulant medication not identified 
1863 
during initial medical screening (including aspirin) will be withdrawn by the PI in 
1864 
consultation with OMSO. Volunteers will be instructed about precautions against 
1865 
hematoma and infection.  They will be given a handout outlining instructions for proper 
1866 
care of the incision site (see “Biopsy Care Instructions”).  Muscle biopsies will be 
1867 
performed using sterile procedures by Dr. Stefan Pasiakos or Dr. Lee Margolis, who will 
1868 
abide by USARIEM’s Percutaneous Skeletal Muscle Biopsy SOP (OMSO-approved 
1869 
USARIEM SOP for Invasive Procedures, Chapter 10) of 11 July 2017 in all regards. The 
1870 
PI and OMSO will follow-up with volunteers within 3 d post-biopsy to monitor for any sign 
1871 
of infection, bleeding, or hematoma. 
1872 
 
1873 
Research Procedure Name:  Lidocaine Injection 
1874 
Research Procedure Description:  Approximately 8-10 mL of 1% lidocaine will be 
1875 
injected using a 25 g needle at the site of the incision, superficially (i.e., skin) and within 
1876 
the vastus lateralis. 
1877 
Research-related Risks:  Slight pain at the site of injection might occur.  Although rare, 
1878 
anaphylactic reactions may also occur following administration of lidocaine.  Unlikely, but 
1879 
possible side effects could include: dizziness, confusion, shakiness, visual changes, 
1880 
nausea, and unusually slow heartbeat.   
1881 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Measures to Minimize Risks of Harm:  Volunteers will be instructed to notify a study 
1882 
investigator or the project coordinator immediately if an allergic (i.e., swelling, itching, 
1883 
rash, hives, difficulty swallowing, or difficulty breathing) reaction occurs. In the case of 
1884 
severe reaction, lidocaine use will be discontinued and OMSO will be notified 
1885 
immediately.  Dr. Pasiakos or Dr. Margolis will be the only ones administering the 
1886 
lidocaine, and medical staff will be onsite.  The PI and study staff will closely monitor the 
1887 
volunteers throughout the procedure.  
1888 
 
1889 
Research Procedure Name:  Exercise 
1890 
Research Procedure Description:  Exercise includes peak aerobic capacity and 
1891 
glycogen normalization studies on a cycle ergometer, outdoor load carriage exercise, 
1892 
steady-state elliptical and cycle ergometer exercise sessions, exercise associated with 
1893 
the Warfighter tasks.   
1894 
Research-related Risks:  Exercise is generally considered safe and beneficial for 
1895 
individuals without cardiovascular disease. The US prevalence of fatal events is 
1896 
approximately 1:100,000 to 1:300,000 in competitive high school athletes and increases 
1897 
to 1:15,000 to 1:50,000 in athletes over the age of 35.  Current civilian and military 
1898 
guidelines state that individuals less than 40 years of age who have no symptoms of or 
1899 
known presence of heart disease or major coronary risk factors have a low risk for 
1900 
cardiac complications during vigorous exercise.  All volunteers in this study fall into this 
1901 
low risk category.  Local muscle discomfort and fatigue may occur in active muscles 
1902 
during and shortly after exercise.  Exercise often carries a risk of injury, including 
1903 
dehydration, acute musculoskeletal strains and sprains, overuse injuries, and accidental 
1904 
injuries caused by the test apparatus.  The risk of musculoskeletal injury from bouts of 
1905 
endurance exercise is minimal. Muscle soreness, ranging in intensity from mild to 
1906 
severe, may persist for 1 to 7 days. Additional risks associated with exercise include foot 
1907 
blisters, skin chafing, muscle cramps, stress fracture, trauma due to falling, and 
1908 
hypotension following the completion of the exercise bout.  The frequency of many of 
1909 
these risks increases with the duration of the exercise bout.   
1910 
Measures to Minimize Risks of Harm:  Studies have confirmed the safety of maximal 
1911 
exercise testing, particularly among apparently healthy persons without significant 
1912 
cardiovascular risk factors.  As a precaution, there will be at least one spotter/monitor 
1913 
during all exercise sessions, and heart rate will be monitored in real time during testing. 
1914 
Exercise monitors and test administrators will be CPR-certified.  Additional safeguards 
1915 
taken to minimize risk during exercise include: (a) qualified personnel will administer the 
1916 
maximal exercise tests, (b) the volunteer will be asked to report any pain or discomfort 
1917 
resulting from exercise, followed up if necessary by medical examination and 
1918 
postponement or curtailment of further testing, (c) volunteers will be required to wear 
1919 
correctly sized footwear, and (d) volunteers will have access to water (ad libitum) to 
1920 
remain hydrated.  
1921 
 
1922 
Research Procedure Name:  Energy Balance and Negative Energy Balance Diet 
1923 
Interventions 
1924 
Research Procedure Description:  Volunteers will be fed adequate energy to maintain 
1925 
body mass during SUSOPS BAL and only 45% of total energy expenditure during 
1926 
SUSOPS NEG BAL to elicit negative energy balance (where dietary intake is < energy 
1927 
expenditure). Combat rations will be the primary food source (some perishable whole-
1928 
foods).     
1929 
Research-related Risks:  The foods and MRE components used in this study pose no 
1930 
known risks to volunteers. All of the meals that volunteers will be fed during both 96 hour 
1931 

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SUSOPS periods consist solely of MRE components.  Sudden changes to the diet can 
1932 
cause gas, cramping, bloating, constipation, or other abdominal discomfort in some 
1933 
individuals. The main discomfort associated with a low energy diets is hunger. 
1934 
Volunteers will be shown copies of study menus and food lists at the initial study 
1935 
recruitment brief. This will be used to determine if prospective volunteers have an 
1936 
allergy, intolerance, or personal preference to foods listed. 
1937 
Measures to Minimize Risks of Harm:  All efforts will be made to accommodate the 
1938 
volunteers with regard to dietary preferences while keeping the major constituents of the 
1939 
diets consistent with study design and between volunteers. Those who have an allergy 
1940 
or intolerance to a menu component, which cannot be accommodated, will not be 
1941 
enrolled in the study. 
1942 
 
1943 
Research Procedure Name:  Dual energy X-ray absorptiometry (DEXA) Scan 
1944 
Research Procedure Description:  Volunteer will lay face-up on the DEXA 
1945 
densitometer table in shorts, t-shirts, and stocking feet.  Volunteers will be asked to 
1946 
remain motionless for the 8-10 min scan. 
1947 
Research-related Risks:  The DEXA scan is an X-ray and is considered to be a low risk 
1948 
procedure.  The radiation dose of the whole-body DEXA scan is 0.1 mrem.  This dose is 
1949 
equivalent to approximately 1/250 of normal annual background radiation, 1/9 of the 
1950 
radiation received in a transatlantic flight, or 1/30 of the radiation received in a chest X-
1951 
ray.   
1952 
Measures to Minimize Risks of Harm:  A quality assurance check will be completed on 
1953 
the DEXA each day prior to its use; the software will not allow the use of the DEXA 
1954 
densitometer if the quality assurance check fails.   
1955 
 
1956 
**There are no risks associated with bio-electrical impedance body composition 
1957 
measures** 
1958 
 
1959 
**There are no risks associated with saliva sampling measures** 
1960 
 
1961 
Research Procedure Name:  Multi-sugar absorption test for intestinal permeability 
1962 
measurement 
1963 
Research Procedure Description: Participants will consume 2 g sucralose and 2 g 
1964 
erythritol dissolved in 180 mL water. All urine produced over the subsequent 24hr will be 
1965 
collected. 
1966 
Research Related Risks: Sucralose and erythritol are commonly consumed sugar 
1967 
substitutes which may cause gas, cramping, diarrhea or bloating in some individuals.  
1968 
Measures to Minimize Risks of Harm: Any participant reporting gastrointestinal 
1969 
distress following the test will be given the option of not participating in the test at the 
1970 
next opportunity.  
1971 
 
1972 
Research Procedure Name:  SmartPill 
1973 
Research Procedure Description: The SmartPill is an ingestible FDA-approved 
1974 
wireless motility capsule that transits the gastrointestinal tract while continuously 
1975 
measuring pH, temperature, pressure, and gastrointestinal transit time.  A single pill will 
1976 
be ingested by volunteers and allowed to pass normally through the gastrointestinal 
1977 
tract. 
1978 
Research Related Risks: Choking, aspiration, retention gastrointestinal tract.  
1979 
Measures to Minimize Risks of Harm: Potential participants with contraindications for 
1980 
use will not be allowed to participate in the SmartPill procedure.  If they are randomly 
1981 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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selected for SmartPill testing, an alternate will be chosen to participate instead. Pills will 
1982 
be ingested under staff supervision. 
1983 
 
1984 
C6.2 Incidental or Unexpected Findings   
1985 
 
1986 
Health problems identified during the screening process will be documented and a copy 
1987 
provided to the volunteer. The volunteer will be encouraged to make an appointment 
1988 
with their primary care provider for a full evaluation of the problem. Volunteers with 
1989 
evidence of any physical, mental, and/or medical conditions that would make the 
1990 
proposed studies relatively more hazardous will be excluded. 
1991 
 
1992 
C6.3 Potential Benefits   
1993 
 
1994 
There is no direct health or other benefits related to participation in this study. 
1995 
 
1996 
C7.  DATA AND SAFETY MONITORING   
1997 
 
1998 
C7.1 Monitoring 
1999 
 
2000 
The PI will, with the assistance of Associate Investigators and project coordinator, 
2001 
continuously evaluate recruitment, the informed consent process, adverse events, and 
2002 
protocol adherence and deviations in order to identify unanticipated problems or risks to 
2003 
the volunteers associated with the research. The PI will ensure that the number of 
2004 
volunteers recruited for this study complies with the protocol.  The PI will submit a 
2005 
monthly summary of all adverse events to the Research Monitor to determine whether 
2006 
the number of adverse events is excessive for the risks outlined in the research protocol.  
2007 
The PI and onsite physician or PA will discuss “discontinuation criteria” for individual 
2008 
volunteers as the study progresses, based on their observations of the volunteer during 
2009 
testing or non-testing periods. Every morning, volunteers will be asked the following 
2010 
questions to evaluate their readiness to test. 
2011 
 
2012 
 
How have you been feeling well since the last test in our laboratory (below 
2013 
average, average, above average)? 
2014 
 
Do you have any pain or symptoms to report that may affect our testing today 
2015 
(e.g., sinus congestion, fatigue, muscle soreness, fever, gastrointestinal pain, 
2016 
etc.)? 
2017 
 
Have you reported all food and beverages consumed in the last 24 h that 
2018 
were not provided to you by study staff? 
2019 
 
What time did you fall asleep last night and awake this morning? 
2020 
 
What type of exercise or physical activities have you performed in the last 24 
2021 
h?  
2022 
 
2023 
C7.2 Research Monitor 
2024 
 
2025 
The research monitor for this study is MAJ Robin Cushing. This individual is an 
2026 
appropriate subject matter expert not associated with the protocol. The research monitor 
2027 
shall, at a minimum, review all unanticipated problems involving risk to subjects or 
2028 
others, serious adverse events and all subject deaths associated with the protocol and 
2029 
provide an unbiased written report of the event.  Other responsibilities may be assigned 
2030 
by the USA MRDC IRB as needed. 
2031 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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2032 
C8.  REPORTABLE EVENTS    
2033 
 
2034 
C8.1 Expected adverse events 
2035 
 
2036 
An adverse event is defined as any untoward or unfavorable medical occurrence in a 
2037 
human research volunteer, including any abnormal sign (e.g., abnormal physical exam 
2038 
or laboratory finding), symptom, or disease, temporally associated with the individual’s 
2039 
participation in the research, whether or not considered related to the individual’s 
2040 
participation in the research. 
2041 
 
2042 
A Serious Adverse Event is defined as any adverse event temporally associated with the 
2043 
subject’s participation in research that is fatal, life-threatening, permanently disabling, 
2044 
requires inpatient hospitalization, or results in congenital anomalies/birth defect, 
2045 
overdose or cancer, or based on appropriate medical judgment, may jeopardize the 
2046 
volunteer, or may require medical or surgical intervention to prevent one of the above 
2047 
outcomes. 
2048 
 
 
2049 
All medical events that the USARIEM Office of Medical Support and Oversight (OMSO) 
2050 
evaluates will be reported to the ORQC.  The PI will report all adverse events to the 
2051 
Research Monitor.   
2052 
 
2053 
Expected adverse events which are not serious are reported to the IRB at the time of 
2054 
continuing review of the protocol.  These events include bruising, infection, swelling and 
2055 
slight pain from the IV placement; slight pain from the lidocaine injection; pain, soreness, 
2056 
infection, and bruising from the muscle biopsy; feeling faint with IV placement, blood 
2057 
draw or biopsy; fatigue and muscle soreness from study exercises and SUSOPS 
2058 
activities; hunger, bloating, gas, cramping, constipation from the dietary invention; 
2059 
fatigue and headaches during the negative energy balance portion of the dietary 
2060 
intervention. 
2061 
 
2062 
C8.2 Unexpected adverse events and unanticipated problems 
2063 
 
2064 
A serious adverse event is any adverse event temporally associated with the subject’s 
2065 
participation in research that is fatal, life-threatening, permanently disabling, requires 
2066 
inpatient hospitalization, or results in congenital anomalies/birth defect, overdose or 
2067 
cancer, or based on appropriate medical judgment, may jeopardize the participant, or 
2068 
may require medical or surgical intervention to prevent one of the above outcomes. 
2069 
 
2070 
All medical events will be reported to USARIEM’s Office of Medical Support and 
2071 
Oversight (OMSO). OMSO staff will retain a copy of the report in the subject’s OMSO 
2072 
medical file as a means of tracking and analyzing trends in medical events.  The PI will 
2073 
report all adverse events to the Research Monitor, if one was appointed for the study. 
2074 
 
2075 
All unanticipated problems involving risk to subjects or others, and serious adverse 
2076 
events that are unexpected and determined to be at least possibly or definitely related to 
2077 
study participation, will be promptly reported within one working day by phone (508-233-
2078 
6306/4811) or email (usarmy.natick.medcom-usariem.mbx.usariem-rqc@mail.mil) to the 
2079 
USARIEM ORQC and the Commander.  These events will also be reported to the HQ 
2080 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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USAMRDC IRB within one working day by phone (301-619-6240), or by e-mail 
2081 
(usarmy.detrick.medcom-usamrmc.other.irb-office@mail.mil). 
2082 
 
2083 
Adverse events assessed by the PI as not serious and serious adverse events that are 
2084 
deemed to be unrelated to participation in the study will be reported to the IRB at the 
2085 
time of continuing review of the protocol. 
2086 
 
2087 
The research monitor is required to review all unanticipated problems involving risk to 
2088 
volunteers or others, serious adverse events and all volunteer deaths associated with 
2089 
the protocol and provide an unbiased written report of the event.  At a minimum, the 
2090 
research monitor should comment on the outcomes of the event or problem, and in the 
2091 
case of a serious adverse event or death, comment on the relationship to participation in 
2092 
the study.  The research monitor should also indicate whether he or she concurs with the 
2093 
details of the report provided by the study investigator.  Reports for events determined 
2094 
by either the investigator or research monitor to be: possibly related, unexpected, and 
2095 
serious or suggest that the research places subjects or others at increased risk of harm 
2096 
during participation will be promptly forwarded to the ORQC and HQ USAMRDC IRB. 
2097 
 
2098 
In the event of a medical emergency at facilities on the Natick Soldier Systems Center, 
2099 
the local installation emergency management will be contacted immediately by dialing 
2100 
x5911. The installation security personnel will direct the ambulance to the proper 
2101 
location on the installation. While awaiting their arrival, Basic Life Support will be 
2102 
rendered by study personnel or on-site medical coverage. EMS response time to 
2103 
USARIEM is approximately 5 minutes. Transport time to definitive care is approximately 
2104 
8 minutes.  
2105 
 
2106 
C8.3 Adverse device effects: N/A 
2107 
C8.4 FDA-regulated research under IND and IDE: N/A 
2108 
 
2109 
 
2110 
SECTION D:  REFERENCES 
2111 
References 
2112 
 
2113 
1. 
Pasiakos, S.M., et al., Effects of exercise mode, energy, and macronutrient interventions on 
2114 
inflammation during military training. Physiol Rep, 2016. 4(11). 
2115 
2. 
Ostrowski, K., et al., Pro- and anti-inflammatory cytokine balance in strenuous exercise in 
2116 
humans. J Physiol, 1999. 515 ( Pt 1): p. 287-91. 
2117 
3. 
Ostrowski, K., et al., A trauma-like elevation of plasma cytokines in humans in response to 
2118 
treadmill running. J Physiol, 1998. 513 ( Pt 3): p. 889-94. 
2119 
4. 
Steensberg, A., et al., IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans. Am J Physiol 
2120 
Endocrinol Metab, 2003. 285(2): p. E433-7. 
2121 
5. 
Fischer, C.P., Interleukin-6 in acute exercise and training: what is the biological relevance? Exerc 
2122 
Immunol Rev, 2006. 12: p. 6-33. 
2123 
6. 
Chan, M.H., et al., Altering dietary nutrient intake that reduces glycogen content leads to 
2124 
phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene 
2125 
transcription during contraction. FASEB J, 2004. 18(14): p. 1785-7. 
2126 
7. 
Keller, C., et al., Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: 
2127 
influence of muscle glycogen content. FASEB J, 2001. 15(14): p. 2748-50. 
2128 
8. 
Pal, M., M.A. Febbraio, and M. Whitham, From cytokine to myokine: the emerging role of 
2129 
interleukin-6 in metabolic regulation. Immunol Cell Biol, 2014. 92(4): p. 331-9. 
2130 
9. 
Steensberg, A., et al., Interleukin-6 production in contracting human skeletal muscle is influenced 
2131 
by pre-exercise muscle glycogen content. J Physiol, 2001. 537(Pt 2): p. 633-9. 
2132 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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10. 
MacDonald, C., et al., Interleukin-6 release from human skeletal muscle during exercise: relation 
2133 
to AMPK activity. J Appl Physiol (1985), 2003. 95(6): p. 2273-7. 
2134 
11. 
Tsigos, C., et al., Dose-dependent effects of recombinant human interleukin-6 on glucose 
2135 
regulation. J Clin Endocrinol Metab, 1997. 82(12): p. 4167-70. 
2136 
12. 
Carey, A.L., et al., Interleukin-6 increases insulin-stimulated glucose disposal in humans and 
2137 
glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase. Diabetes, 2006. 
2138 
55(10): p. 2688-97. 
2139 
13. 
Febbraio, M.A., et al., Interleukin-6 is a novel factor mediating glucose homeostasis during 
2140 
skeletal muscle contraction. Diabetes, 2004. 53(7): p. 1643-8. 
2141 
14. 
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whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
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Date: 18 July 2019 
 
 
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Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
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v21OCT14 
 
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2350 

Protocol Title: Effects of severe negative energy balance on inflammation, iron absorption, nutritional status, skeletal muscle and 
whole-body metabolic homeostasis, cognitive, and physical performance during a simulated sustained operation (SUSOPS) 
Version # 7   
Date: 18 July 2019 
 
 
HQ USAMRDC IRB Research Protocol Template 
v21OCT14 
 
Page 59 of 59 
 
108. 
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2351 
appetite control. Sports Med, 2015. 45(5): p. 659-78. 
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2358 
 
2359 
 
2360 
 
2361 
SECTION E:  ABBREVIATIONS AND ACRONYMS 
2362 
ACSM, American College of Sports Medicine; BART, balloon analogue risk test; BAL, energy 
2363 
balance; EVAR, evaluation of risks scale; DEXA, dual energy x-ray absorptiometry; DLW, 
2364 
doubly labeled water; GLP-1, glucagon-like peptide-1; IL-6, interleukin 6; LFPQ, Leeds Food 
2365 
Preference Questionnaire; NEG BAL, MRE, Meals Ready-to-Eat; negative energy balance; PP, 
2366 
pancreatic polypeptide; POMS, profile of mood states; PVT, psychomotor vigilance test; PYY, 
2367 
peptide-YY; RMR, resting metabolic rate; SIgA, Secretory IgA; SUSOPS, sustained operations; 
2368 
TBW, total body water; TDEE, total daily energy expenditure; TNF-α, tumor necrosis factor 
2369 
alpha; VAS, visual analogue scale; VO2peak, peak oxygen consumption; refer to table 1 for 
2370 
appropriate definitions for analyte abbreviations.  
2371 
 
2372 
 
2373 
 
2374 
SECTION F: DoD PRIVACY RULE AND PROTECTED HEALTH INFORMATION (HIPAA) 
2375 
Click in the appropriate box See the “Guide for Investigators” for definitions and further 
2376 
information.   
2377 
 
2378 
 NA – institution is not a covered entity 
2379 
 
2380 
 NA – will not use or disclose protected health information 
2381 
 
2382 
 HIPAA authorization will be obtained  
2383 
 
2384 
 An application for waiver/alteration of HIPAA authorization will be submitted   
2385 
 
2386