The Effect of Impact Loading on Bone Biomarkers in Energy-Restricted Female Runners
Phase: NA
Conditions: Relative Energy Deficiency in Sport
Sponsor: Virginia Polytechnic Institute and State University

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INSTRUCTIONS: 
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Use this “TEMPLATE PROTOCOL (HRP-503)” to prepare a study protocol
outlining your research plan.
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you can mark as “N/A”if you are certain that the subsection is not applicable.
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create a new paragraph wherever you need to make a change, and indicate
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management will store the older versions of your protocol if the IRB or HRPP
staff need to compare them during the review.
PROTOCOL TITLE: 
Include the full protocol title. 
The Effect of Impact Loading on Bone Biomarkers in Energy-Restricted Female 
Runners 
PROTOCOL NUMBER:  
Include the number assigned in Protocol Management (verify this has been added 
before submitting protocol to HRPP).  
22-168
PRINCIPAL INVESTIGATOR: 
Full Name and Degrees: Dawnine Enette Larson-Meyer, PhD 
Department: Human Nutrition, Foods, and Exercise 
Telephone Number: 540-231-1025 
Email Address: enette@vt.edu 
FUNDING: 
Sponsor(s): no sponsor 
Funded already or in the proposal phase?: Proposal phase 
Is Virginia Tech the primary awardee or the coordinating center of this grant or 
contract? If not, list the primary institution: N/A 
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VERSION NUMBER/DATE:  
Include the version number and date of this protocol. Versions should start at 1.0. 
 
Version 6.0 
 
REVISION HISTORY:  
Use this table to keep track of changes. Add more rows as needed. 
 
Revision 
# 
Version Date 
Brief Summary of Changes  
(i.e., the different sections) 
Consent 
Change? 
1 
3/1/2022 
Clarified time commitment for subject 
participation and payment structure in the 
protocol and consent form (Sections 1.0, 15.4, 
17.4).  
Added more details regarding future 
undergraduate study personnel in section 26. 
No 
2 
3/16/2022 
Increased compensation to $100 split into two 
payments of $25 and $75. (Section 15.4) 
Clarified exclusion criteria: no contraceptive 
use, no recent (within 12 mo) or current eating 
disorder (Section 12.2) 
Expanded on protocol justification (Section 
3.3) 
Clarified running time of protocol (Section 8.1) 
Yes 
3 
3/29/2022 
Added pregnancy test after the 3-week washout 
period on day 1 of the second experimental 
condition (Sections 8.1, 8.2, 12.3, 17.4) 
Yes 
4 
5/20/2022 
Protocol for impact loading exercise sessions 
modified slightly to include recovery period 
(Section 8.2) 
Additional assays (leptin, cortisol, ferritin, 
25(OH)D, Nesfatin-1) (Section 4.1 and Table 1 
in Section 8) 
Added vitamin D questionnaire at baseline 
(Section 8.2) 
Change to weight safety endpoint (Section 4.2 
and justification in section 7.2) 
Change timepoints for running economy 
Added menstrual cycle question to phone 
screening form 
Yes 
5 
6/24/2022 
Baseline DXA moved from visit 2 to visit 1 
(Section 8.2) 
Yes 
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Added a mealtime log during the 5-day 
experimental phases (Section 8.2) 
Protocol for treadmill run decreased to 
65%VO2max (previously 70%VO2max) 
(Section 8.2) 
6 
9/6/2022 
Inclusion criteria adjusted to include 
participants ages 18-35 (previously 18-30) and 
removed running requirement of 30 miles/week 
and VO2max criteria (Section 12) 
Additional iron status markers (TIBC and 
serum iron) (Section 4.1, Table 1 Section 8) 
Exclusion criteria: criteria for low BMD 
adjusted to z-score <-2 (Section 12) 
Yes 
7 
7/10/2023 
Inclusion criteria adjusted to include 
participants with a BMI 18.5-30 (Section 12) 
Yes 
8 
9/8/23 
Increase sample size to 20 
No 
 
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Table of Contents 
1.0 
Study Summary 
5 
2.0 
Objectives 
6 
3.0 
Background 
6 
4.0 
Study Endpoints 
10 
5.0 
Study Design and Statistical Analysis Plan 
11 
6.0 
Setting 
12 
7.0 
Study Intervention(s)/Investigational Agent(s) 
12 
8.0 
Procedures Involved 
14 
9.0 
Data and Specimen Long Term Storage and Use 
24 
10.0 Sharing of Results with Subjects 
26 
11.0 Study Timelines 
27 
12.0 Inclusion and Exclusion Criteria 
27 
13.0 Vulnerable Populations 
29 
14.0 Number of Subjects 
30 
15.0 Recruitment Methods 
31 
16.0 Withdrawal of Subjects 
32 
17.0 Risks to Subjects 
33 
18.0 Potential Benefits to Subjects 
36 
19.0 Data Management and Confidentiality 
37 
20.0 Provisions to Protect the Privacy Interests of Subjects 
39 
21.0 Provisions to Monitor the Data to Ensure the Safety of Subjects 
40 
22.0 Compensation for Research Related Injury 
42 
23.0 Economic Burden to Subjects 
42 
24.0 Consent Process 
42 
25.0 Process to Document Consent in Writing 
46 
26.0 Resources Available 
47 
27.0 Multi-Site Research 
49 
 
 
 
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1.0 
Study Summary 
 
Study Title 
The Effect of Impact Loading on Bone Biomarkers in 
Energy-Restricted Female Runners 
Study Design 
Randomized Cross-Over Intervention Study 
Primary Objective 
Determine the effect of short-term, high-impact loading on 
biomarkers of bone remodeling in trained, female long-
distance runners in the presence of low energy availability. 
Secondary 
Objective(s) 
Exploratory Aim: Examine the relationship between 
undercarboxylated osteocalcin and impact loading on 
glucose metabolism during acute low energy availability. 
Study Population 
Eumenorrheic, female long-distance runners who do not 
have existing low energy availability. 
Sample Size 
20 
Research 
Intervention(s)/ 
Investigational 
Agent(s)  
Research volunteers will undergo two experimental 
conditions of low energy availability in a randomized order: 
Low energy availability with daily running only (RUN) and 
low energy availability with daily running and impact 
loading (RUN+IL). Each 5-day experimental condition will 
be initiated in the follicular phase and separated by a 
washout period of one menstrual cycle (approximately 24 
days). 
Study Duration for 
Individual 
Participants 
19 days of intervention and study visits including one 
screening visit, one baseline testing visit, 3 days of tracking 
normal diet and exercise, and two experimental conditions of 
7 days separated by a 3-week washout period of normal diet 
and exercise  
Acronyms and 
Definitions  
ACSM, American College of Sports Medicine; BMD, bone 
mineral density; CBC, complete blood count; CGM, 
continuous glucose monitoring; CTX, C-terminal telopeptide 
of type 1 collagen; DXA, Dual-Energy X-ray 
Absorptiometry; HIP Laboratory, Human Integrated 
Physiology Laboratory; LEA, low energy availability; NEM 
Laboratory, Nutrition and Exercise Metabolism Laboratory; 
P1NP, N-terminal propeptide of type 1 procollagen; Trap5b, 
tartrate-resistant acid phosphatase; TSH, thyroid-stimulating 
hormone; unOC, undercarboxylated osteocalcin; VO2 max, 
maximal oxygen uptake during exercise, also known as 
aerobic capacity;  
 
 
 
 
 
 
 
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2.0 
Objectives 
2.1 Describe the purpose, specific aims, or objectives of this study: 
1) Determine the effect of short-term, high-impact loading on biomarkers of bone 
remodeling in energy-restricted, female long-distance runners. 
2) Examine the relationship between undercarboxylated osteocalcin and impact 
loading on glucose metabolism during acute low energy availability. 
 
2.2 State the hypotheses to be tested:  
The addition of 50 high-intensity impact loading jumping exercises per 
day to usual run training will result in less suppression of bone formation 
following 5 days of endurance running in an energy-restricted condition 
compared to daily running in an energy-restricted state without high-
impact loading exercises.  
 
3.0 
Background 
3.1 Summarize the relevant prior research on this topic and gaps in current 
knowledge within the field of study: 
 
Maintaining adequate energy intake is essential for athletic performance and 
overall general health. Yet, many athletes experience low energy availability (LEA) by 
failing to consume enough calories to meet their energy demands. Energy availability is 
the amount of energy remaining from dietary energy intake (EI) to support general health 
and bodily functions after accounting for exercise energy expenditure (EEE), and is 
commonly expressed relative to fat-free mass as kilocalories per kilogram of fat-free 
mass (FFM) per day (kcal/kgFFM/d). According to the Life History Theory, the human 
body will adapt to conserve energy under conditions of biological stress, such as energy-
restriction and LEA, by downregulating biological processes that are less essential for 
immediate survival (Shirley, Long et al. 2022).  The performance and health 
consequences of LEA are characterized by the syndrome known as Relative Energy 
Deficiency in Sport (RED-S) (Mountjoy, Burke et al. 2018). Specific to this proposed 
study, athletes suffering from RED-S and chronic LEA may experience several inter-
related health consequences involving endocrine function, reproduction, and bone 
metabolism. Previous studies indicate LEA is associated with increased risk for bone 
stress injuries and low bone mineral density (BMD) (Tenforde, Carlson et al. 2016, 
Tenforde, Carlson et al. 2018, Gibbsm Battuv et ak, 2014), in addition to suppression of 
certain bone-regulating hormones including estrogen, insulin, vitamin D, triiodothyronine 
(T3), and insulin-like growth factor (IGF-1) (McCall, Ackerman 2019).  
 
Previous Studies on LEA and Biomarkers of Bone Metabolism. Biomarkers of 
bone formation and resorption are often used as surrogate markers for assessing bone 
metabolism in the short term given that it can take months or even years for changes in 
bone microarchitecture and BMD to manifest. In a study of 8 male distance runners, 
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serum N-terminal pro-peptide of type 1 collagen (P1NP), a marker of bone formation, 
and IGF-1 decreased by 15% and 17%, respectively, after 3 consecutive days of treadmill 
running with energy intake restricted to 50% of estimated needs (Zanker and Swaine 
2000). In a more recent cross-over design study (Papageorgiou, Elliott-Sale et al. 2017), 
changes bone biomarkers were measured in 11 women and 11 men in response to 5 days 
of daily running under optimal EA conditions (45 kcal/kgFFM/d) and 5 days of daily 
running under low EA conditions (15 kcal/kgFFM/d). In both conditions, participants ran 
on a treadmill until an energy expenditure of 15 kcal/kgFFM was achieved. Diets 
provided 60 kcal/kgFFM/d and 30 kcal/kgFFM/d for the optimal and low EA conditions, 
respectively. In the female participants, P1NP was significantly lower after the low EA 
condition compared to optimal EA. Additionally, ß-carboxyl-terminal cross-linked 
telopeptide of type 1 procollagen (ß-CTX), a marker of bone resorption, increased 19% in 
response to the low EA condition and this increase was significantly different from the 
change in ß-CTX observed in the optimal EA condition. No significant change in P1NP 
or ß-CTX were observed in men. This suggests women may be more sensitive to the 
effects of LEA on bone metabolism and, therefore, be at greater risk. In another cross-
over design study of 10 female runners, changes in P1NP and ß-CTX were measured 
after completion of three, 3-day experimental conditions: optimal EA (45 kcal/kgFFM/d), 
LEA achieved through dietary restriction only (15 kcal/kgFFM/d), and LEA achieved 
through a combination of dietary restriction and daily running (15 kcal/kgFFM/d) 
(Papageorgiou, Martin et al. 2018). In the combined diet and exercise LEA condition, 
participants received diets providing 30 kcal/kgFFM/d and expended 15 kcal/kgFFM/d 
during a treadmill run. This study found P1NP was significantly reduced after 3 days of 
LEA achieved through diet only, but not after the combined LEA condition with running. 
These findings suggest the negative effects of LEA on bone metabolism may be 
counteracted or masked by the osteogenic effects of weight-bearing exercise, such as 
running. There were no significant changes observed in ß-CTX in response to either LEA 
condition. Based on these short-term studies, it appears suppression of bone formation 
occurs before measurable increases in bone resorption. Additionally, the severity of bone 
impairments may depend on the degree of energy restriction given the dose-response 
relationship observed between LEA and select bone turnover markers in exercising 
women (Ihle and Loucks 2004).  
 
Benefit of High-Impact Loading on Bone Metabolism. High-impact loading 
exercises such as jumping, bounding, and plyometric training place a high level of 
mechanical strain on bone and can elicit osteogenic adaptations (Hutson, O’Donnell et al. 
2021). There have been a limited number of studies showing mixed results on the short-
term effects of high-impact loading on bone biomarkers (Rantalainen, Heinonen et al. 
2009, Rogers, Dawson et al. 2011). Additionally, very few studies have included energy 
intake assessments and most studies have been conducted in non-athletes. In one study of 
26 female non-athletes, markers of bone formation (osteocalcin and bone specific 
alkaline phosphatase (BAP)) and bone resorption (tartrate-resistant acid phosphatase 
(TRAP5b) and CTX) were measured in response to either a control condition or jumping 
intervention (Kishimoto, Lynch et al. 2012). Volunteers in the jump group performed 10 
jumping exercise per day at a frequency of 5 times a week for 2 weeks. Bone resorption 
measured by CTX was lower in the jump group compared to baseline. Additionally, 
TRAP5b was significantly lower in the jump group compared to the control group, 
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however, there was no significant changes in TRAP5b from baseline in either group. 
Interestingly, the jump intervention significantly lowered BAP, a marker of bone 
formation. No changes were observed in osteocalcin within or between groups. This 
study was limited based on lack of energy intake assessment and the potential for inter-
subject variability in bone response to jumping exercises. Despite the theoretical basis for 
high-impact exercise as a countermeasure for bone resorption during LEA, there have 
been no controlled trials to date that have investigated this theory in energy-restricted 
athletes. 
Undercarboxylated Osteocalcin (unOC) and Glucose Metabolism. This study 
seeks to contribute to understanding the potential association between osteocalcin in 
glucose metabolism in humans. Undercaboxylated osteocalcin (unOC) is the active form 
of osteocalcin and is released in response to osteoclast activity during bone resorption 
(Moser and van der Eerden 2019). The role of unOC to regulate glucose metabolism in 
humans is unclear (Lin, Brennan-Speranza et al. 2018). In a cross-sectional study, unOC 
was found to be associated with insulin secretion and sensitivity in lean male patients 
(BMI <25 kg/m2) (Fernández-Real, Izquierdo et al. 2009). Subjects in this study were 
generally healthy and non-athletes. In a meta-analysis of osteocalcin and glucose 
metabolism, a weak negative correlation (r=-0.09, p<0.5) was found between unOC and 
fasting plasma glucose in women (Liu, Guo et al. 2015). However, the association 
between unOC and glucose metabolism was found to be stronger in men than in women.  
There is limited data on the effects of LEA on glucose metabolism in athletes without 
diabetes. One study of 7 male long-distance runners found muscle glycogen was reduced 
by approximately 30% after 3 days of endurance training under energy-restricted 
conditions (EA = 18.9 ± 1.9 kcal/kgFFM/d) (Kojima, Ishibashi et al. 2020). No studies to 
date have investigated the relationship between undercarboxylated osteocalcin and 
glucose metabolism in energy-restricted athletes. 
 
 
3.2 Describe any relevant preliminary data: 
N/A 
 
3.3 Based on the existing literature, provide the scientific or scholarly 
rationale for and significance of your research and how will it add to 
existing knowledge: 
Maintaining adequate energy intake is essential for athletic performance 
and general health. Yet, many athletes experience low energy availability 
(LEA) by failing to consume enough calories to meet their energy 
demands. Recent studies estimate that the prevalence of LEA in athletes 
ranges from 22% to 58% with a high prevalence in women and endurance 
sports (Logue, Madigan et al. 2020). Chronic LEA is associated with 
several inter-related health consequences involving endocrine function, 
reproduction, and bone metabolism. Impaired bone health is one of the 
most concerning consequences of chronic energy restriction. Left 
untreated, prolonged energy deficiency may impair bone accrual during 
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adolescence and bone formation in adulthood, leading to an elevated risk 
of fracture and osteoporosis later in life (Papageorgiou, Dolan et al. 2018). 
The recommended treatment for LEA is to increase energy availability to 
optimal levels (45 kcal/kgFFM/d) by increasing EI, decreasing EEE, or a 
combination of the two (Kuikman, Mountjoy et al. 2021). However, 
inadequate energy is not always intentional as in the cases of disordered 
eating/eating disorders and “cutting weight”. Some athletes may 
experience LEA unintentionally due to factors such as inadequate 
knowledge of fueling recommendations, decreased appetite, lack of time, 
or low food security (Wasserfurth, Palmowski et al. 2020). Therefore, not 
all athletes may be willing or able to achieve optimal energy availability. 
Given the significant risk to long-term bone health, strategies to counteract 
the effects of LEA on bone metabolism are necessary.  
Compared to inactive controls, runners on average have higher BMD and 
bone strength (Scofield, Hecht et al. 2012). However, lower total and site-
specific BMD has been reported in female endurance runners compared to 
sprinters and athletes competing in higher impact sports (Tenforde, 
Carlson et al. 2018, Mudd, Fornetti et al. 2007). Lower BMD in this 
population may partially be attributed to a high prevalence of LEA and 
risk of disordered eating (DE) and clinical eating disorders (EDs) among 
female and endurance athletes (Melin, Tornberg et al. 2015). Mechanical 
loading through weight-bearing exercises, such as high-impact loading 
activities and resistance training, provides an osteogenic stimulus and non-
pharmacological approach to improving bone health (Hart, Nimphius et al. 
2017, Beck, Daly et al. 2017). In a cross-sectional study of male distance 
runners, BMD was found to be significantly higher in runners who 
reported routine engagement in resistance training compared to runners 
who did not weight train and untrained controls (Duplanty, Levitt et al. 
2018).  
Despite the advantages of resistance training on bone health, 
recommending additional resistance training to athletes with LEA may 
further exacerbate energy deficiency if EI is not increased. Thus, physical 
activity interventions to counteract LEA must be designed to achieve 
maximal osteogenic responses with the minimal possible energy cost. 
Adding additional exercise without EI compensation may worsen the state 
of energy deficiency can cause further damage to the athlete’s health. 
Even if an athlete does increase EI, adherence to additional routine 
resistance training is another potential issue. A survey of 667 competitive 
distance runners found only 60% of respondents engaged in routine 
resistance training, with middle-distance (800-3,000 m) runners reporting 
higher participation in strength and conditioning activities compared to 
long-distance (5k to half-marathon) runners (Blagrove, Brown et al. 2020). 
Middle-distance runners were 2.7 and 6.7 times more likely to engage in 
resistance training compared to long-distance and ultra-distance runners, 
respectively. An alternative to approach to resistance training that would 
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also apply mechanical loading to the bone is high-impact loading exercises 
like jumping and plyometric training. Brief jumping exercises have the 
potential to cause a significant osteogenic bone response with very little 
energy expenditure, given that a relatively low volume of 10-50 
impacts/day at a frequency of 4-7 days/week is required to produce 
osteogenic effects in premenopausal women (Kishimoto, Lynch et al. 
2012, Bailey and Brook-Wavell et al. 2010). The protocol in this proposed 
study will use jumping exercises at a volume of 5 sets of 10 jumps each 
day (50 jumps/day) with a 60 second rest between each set. Jumps will be 
at an intensity of 2x body weight and performed on 5 consecutive days. 
This approach has several benefits for runners with LEA, with the first 
being a relatively low energy cost of the jumping intervention which will 
prevent worsening the LEA state. The second benefit is the short time 
commitment required for high-impact loading exercises. Given the high 
running volume of most long-distance runners, engagement in additional 
cross-training such as resistance exercises may be a challenge. Thus, 
athletes may find it easier to adherence to the jumping exercises proposed 
in this study compared to other exercise interventions that require more 
time and effort. Based on the elevated risk of LEA and low BMD in 
female endurance runners, this population would benefit from this 
proposed study. 
Findings from this proposed study will be of interest to sports dietitians 
and athletic trainers based on its potential to improve the clinical 
management of bone loss in female athletes with LEA through the use of 
brief, high-impact loading exercises. Future trials based on findings of the 
proposed study will likely explore the long-term efficacy of high-impact 
loading during prolonged LEA in active individuals across the spectrum of 
physical activity levels. 
 
4.0 
Study Endpoints 
4.1 Describe the primary and secondary study endpoints. See links below for 
discussion of study endpoints and how they may differ from study 
objectives. These are most common in clinical trials but are sometimes 
applicable to other types of biomedical research, as well as social, 
behavioral, or educational research. See link below for a discussion. 
https://docs.google.com/document/d/1Wocz7K7a0hCQJPPO_khh5l1
SQQjhGDDGHzcOPRHR5Tw/edit?usp=sharing  
 
Primary Endpoints 
Change in Biomarkers of Bone Remodeling in Blood including N-terminal 
propeptide of type 1 procollagen, undercarboxylated osteocalcin, sclerostin, and 
C-terminal telopeptide of type 1 collagen. 
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Change in Hormone and Metabolic Markers in Blood including parathyroid 
hormone, estrogen, insulin-like growth factor 1, hepcidin, insulin, cortisol, leptin, 
Nesfatin-1, and thyroid hormones 
24-h Glucose 
Secondary Endpoints 
Change in Running Economy 
Change in Body Weight 
Change in ferritin, TIBC, iron, vitamin D 
 
4.2 Describe any primary or secondary safety endpoints. These should be 
included for all studies that are greater than minimal risk. (Minimal risk: 
The probability and magnitude of harm or discomfort anticipated in the 
research that are not greater in and of themselves than those ordinarily 
encountered in daily life or during the performance of routine physical or 
psychological examinations or tests.): 
 
Safety Endpoints  
Excess weight loss (>5 lbs. during the 5-day experimental conditions) 
Excessive change in absolute value for 24-hour glucose concentration 
Excessive muscle soreness and/or pain 
Fainting or light-headedness 
Muscle or joint injury 
General fatigue 
 
5.0 
Study Design and Statistical Analysis Plan 
5.1 Describe the basic study design/approach (e.g., qualitative study using five 
focus groups of first year students to describe assimilation into the 
university community; randomized controlled trial of a behavioral change 
intervention to increase dietary intake of whole grains; pre- post-test 
evaluation of new pedagogical techniques to improve adult literacy): 
 
This study is a randomized, cross-over intervention study that will evaluate 
the effect of brief, high-impact loading exercises on biomarkers of bone 
metabolism in energy-restricted, eumenorrheic female runners. Volunteers 
will complete two, 5-day experimental conditions in a randomized order 
separated by one menstrual cycle (approximately 3 weeks). Experimental 
conditions will include a dietary intervention of energy intake equal to 30 
kcal/kgFFM/d using controlled diets and an exercise intervention of daily 
treadmill running with or without an additional 50 impact loading exercises. 
 
 
 
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5.2 Describe corresponding data analysis plan/approach (e.g., content analysis 
of focus group transcripts; descriptive analysis followed by linear 
regression modeling; nonparametric analysis of pre- and post-test 
measures): 
 
Data will be analyzed using IBM SPSS statistics software. Paired t-test will be used to 
detect differences in bone biomarkers and hormones within and between experimental 
conditions (Aim 1). Associations between undercarboxylated osteocalcin concentration 
and glucose metabolism (i.e., interstitial glucose concentration and serum insulin) will be 
analyzed using regression analysis (Aim 2). Data will be summarized as mean ± 1 
standard deviation. The significance level will be set a priori at p<0.05. 
 
 
6.0 
Setting 
6.1 Describe the sites or locations where your research team will conduct the 
research.  Consider each of the items listed below: 
● 
Identify where your research team will identify and recruit potential 
subjects. 
● 
Identify where the team will perform the research procedures. 
● 
Describe the composition and involvement of any community 
advisory board(s). 
● 
For research conducted in other locations, describe: 
o 
Site-specific regulations or customs affecting the research at 
those locations. 
o 
Local scientific and ethical review structure at those locations.  
Examples include work in other cultures or ethnic groups 
(within or outside of the U.S.) and work with churches. The 
HRPP will provide additional guidance for international 
research. 
 
 
The research will be conducted at Virginia Tech. Our research team will identify and 
recruit potential research participants and also perform the research procedures in the 
Human Integrative Physiology (HIP) Laboratory in the Garvin Innovation Building, 233 
Wallace Hall, and the Nutrition and Exercise Metabolism (NEM) Laboratory in the 
research building located on 2270 Kraft Drive in the CRC.   
 
 
7.0 
Study Intervention(s)/Investigational Agent(s) 
7.1 Describe the study interventions (including behavioral interventions) and/or 
investigational agents (e.g., drugs or devices) to be used in this study.  Consider 
each of the items listed below:  
● 
Drug/Device Handling: If the research involves drugs or devices, 
describe your plans to store, handle, and administer the drugs or 
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devices so that they will be used only on subjects, and only by 
authorized investigators. 
● 
Describe whether any of the following will be used: microwaves, X-
rays, DEXA scans, general anesthesia, or sedation 
● 
If control of the drugs or devices used in this protocol will be 
accomplished by following an established, approved organizational 
SOP (e.g., Research Pharmacy SOP for the Control of 
Investigational Drugs, etc.), please reference the SOP in this section. 
 
 
The study intervention will consist of controlled meals providing 30 
kcal/kgFFM/d and exercise intervention (running with and without impact 
loading exercises) that will result in energy expenditure of 15 kcal/kgFFM. 
Menus will be designed by a registered dietitian (T.Sterringer) and provide 55% 
of total calories from carbohydrates, 20% from protein, and 25% from fat 
(example diet uploaded). A daily multi-vitamin will be provided during the 
nutrition intervention to ensure micronutrient needs are met during the acute 
energy restriction. 
 
The Research does not involve administration of drugs.  The research does 
involve use of Dual-energy x-ray absorptiometry (DXA/DEXA) scans that will 
be performed at 1 timepoint during the study (baseline). DXA scans will be 
performed by an ISCD Certified Bone Densitometry Technologist (T. 
Sterringer). 
 
 
7.2 List the name of all drugs (including any vitamins, supplements, herbs, or 
nicotine) to be used in the study.  Indicate whether they have FDA 
approval, and list any limitations for their use:  
 
A standard over the counter, multi-vitamin and mineral supplement 
(Nature Made) will be provided during the 5-days of controlled 
experimental diet to ensure micronutrient needs are met in the presence of 
energy restriction. Nature Made multivitamin supplements are verified by 
the United States Pharmacopeia (USP), a nonprofit organization that offers 
third-party verification of product quality and labeling accuracy.  
 
7.3 List all devices, how they will be used, their purpose in the study, and if 
they will be used in a manner consistent with their approved uses. If they 
will be used in ways that are not yet FDA approved, indicate whether they 
need an IDE or a determination that they are exempt from the IDE 
Determination. If a determination of significant risk or non-significant risk 
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is needed for any of the devices, include the researcher’s recommendation 
for each of those devices:  
 
The medical devices/equipment used in this study include the DXA and 
the CGM sensors.  
The DXA will be used to assess total body composition at baseline. Both 
devices are FDA approved and the research will involve employment of 
these devices for approved uses. Scans will be performed only by 
members of the research staff who are trained and certified bone 
densitometry technologists (CBDT) through the International Society of 
Clinical Densitometry.  
The CGM will be used only for research and not diagnostic purposes for 
the intended FDA approved intent of monitoring blood glucose 
concentration over several days. Prescriptions are not required to obtain 
CGM devices from Abbott Laboratories (FreeStyle Libre) if they are to be 
used for research purposes.  Dr. Larson-Meyer has experience using CGM 
for research purposes as part of an ongoing research project (IRB #21-
561).  
7.4 If the drug is investigational (has an IND) or the device has an IDE or a 
claim of abbreviated IDE (non-significant risk device), include the 
following information: 
● 
Identify the holder of the IND/IDE/abbreviated IDE. 
● 
Explain procedures followed to comply with sponsor requirements 
for FDA regulated research for the following: 
 
Applicable to: 
FDA Regulation 
IND Studies 
IDE studies 
Abbreviated 
IDE studies 
21 CFR 11 
X 
X 
 
21 CFR 54 
X 
X 
 
21 CFR 210 
X 
 
 
21 CFR 211 
X 
 
 
21 CFR 312 
X 
 
 
21 CFR 812 
 
X 
X 
21 CFR 820 
 
X 
 
 
N/A 
 
8.0 
Procedures Involved 
8.1 Describe and explain the study design: 
This study will employ a cross-over design in which 12 eumenorrheic women between 
the ages of 18 and 35 will complete two experimental conditions in a randomized order 
using a computer program with a random number generator. The cross-over study design 
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will help control for inter-subject variability in response to LEA (Guebels, Kam et al. 
2014, Reed, De Souza et al. 2015). 
 
As outlined in the table, eligible participants will undergo two, 5-day 
experimental conditions separated by one menstrual cycle (approximately 24 days) 
consisting of an energy-restricted diet and endurance training regimen. Experimental 
conditions will include a dietary intervention of energy intake equal to 30 kcal/kgFFM/d 
using controlled diets and an exercise intervention of daily treadmill running with one 
condition of run only (RUN) and one condition with running and 50 high-intensity, 
impact loading “jumping” exercises (RUN+IL) After providing informed consent, 
participants will be randomly assigned to the RUN or RUN+IL condition. Outcome data 
will include assessment of serum biomarkers of bone metabolism and circulating 
hormones important for bone metabolism. Compliance to the nutrition and exercise 
regimen will be evaluated at regular intervals and include collection of packaging and 
uneaten food at the end of each 5-day condition, measurements of body weight before 
and after each experimental phase to assess change, and physical activity data collected 
by accelerometer and digital technology provided by the research team (Garmin smart 
watch and My PT Hub app).   
 
Dietary Intervention. During the two experimental conditions, participants will be 
provided controlled, weighed diets equal to 30 kcal/kgFFM/d. Energy intake will be 
manipulated individually based on the FFM of participants measured via DXA. Diets will 
be prepared by a registered dietitian (TS) in the research kitchen in Wallace Hall and 
standardized between conditions to include three meals and one snack. Menus will 
consist of similar whole foods and commercial products that provide approximately 55% 
of total calories from carbohydrates, 20% protein, and 25% fat. Diets will be modified 
based on participant allergies or preferences, within reason. Participants will be instructed 
to consume the meals and snack at approximately the same time each day to avoid 
within-day fluctuations in energy balance (Fahrenholtz, Sjödin et al. 2018). They will 
also be instructed not to consume any other foods or beverages other than water and non-
calorie beverages (e.g., black coffee, unsweetened tea). A daily multi-vitamin will be 
provided to participants during the experimental conditions to provide adequate 
micronutrient intake during the energy-restricted state. The key investigators are both 
registered dietitians with experience assessing energy balance. The PI and has extensive 
experience conducting controlled feeding trials.  
 
Exercise Sessions. Participants will undergo supervised exercise sessions 
consisting of treadmill running with and without high-impact exercises on 5 consecutive 
days in the NEM laboratory on two occasions separated by one menstrual cycle 
(approximately 24 days). On the first day of each experimental condition, exercise energy 
expenditure (EEE) will be measured using indirect calorimetry (ParvoMedics) during a 
controlled “titration” run to help determine running speed at 65% VO2max. A heart rate 
monitor will be used simultaneously to measure heart rate. Total EEE for the exercise 
session is approximately 15 kcal/kgFFM. From this, the running protocol (duration) 
needed to expend or “burn” a total of 15 kcal/kgFFM will then be determined and used 
throughout the study. In the RUN condition, participants will run on the treadmill run at 
that pace for the amount of time needed to expend 15 kcal/kgFFM each day of the five-
day intervention. Running time on the treadmill will vary based on participant body 
weight, percent body fat, VO2max, and running efficiency. Treadmill duration will be 
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determined based on an expenditure of 15 kcal/kgFFM while running at an intensity of 
65% VO2max. We estimate total running time for participants to fall within a range of 
approximately 50-65 minutes. For example, a runner with the following characteristics: 
body weight = 62kg, body fat % = 30%, VO2max = 55 ml/kg/min, would need to run for 
58 minutes to meet an energy expenditure of 15 kcal/kgFFM. Runners with a higher 
running efficiency and VO2max will have slightly shorter running durations compared to 
runners who are less efficient. In the RUN+IL condition, participants will start by 
performing 5 sets of 10 high-impact loading jumping movements for a total of 50 
impacts/session at intensities greater than 2x bodyweight with 60 seconds of rest between 
sets to stimulate an osteogenic response according to the guidelines for osteoporosis 
prevention recommended by the Exercise and Sport Science Australia (ESSA) (Beck, 
Daly et al. 2017). Intensity of the jumping exercises will be assessed using ground 
reaction force measured using dual force plates. Participant body weight will be obtained 
before each exercise session to ensure GRF of jumping exercises is at the desired 
intensity. The digital scale and monitor will be separate, and body weight will not be 
shared with the participant. Indirect calorimetry will also be used simultaneously on day 
one of the intervention (only) to assess the energy expended during the jumping 
exercises. Participants will then run on the treadmill at 65% VO2max until combined 
energy expenditure of impact loading and treadmill running reaches the target 15 
kcal/kgFFM. Participants will also wear an accelerometer and smart watch devices during 
the experimental conditions to measure activity level. All activity tracking devices will be 
provided by the research team. Participants will be instructed to refrain from all physical 
activity outside of the supervised exercise sessions that are not related to activities of 
daily living (e.g., getting dressed, walking to the car).  
Table 1. Overview of Data Collection 
Overview of Data Collection 
 
 
 
 
 
 
 
 
 
 
 
RUN and RUN+IL Experimental Conditions 
 
 
Baseline 
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
Day 6 
Day 7 
Body Mass (weight) 
x 
x 
x 
x 
x 
x 
x 
x 
Body Composition by DXA 
x 
 
 
 
 
 
 
 
BMD (total body, dual femur, 
lumbar spine) by DXA 
x 
 
 
 
 
 
 
 
Urine collection for hydration 
status  
x 
 
 
 
 
 
 
 
Pregnancy test 
x 
x* 
 
 
 
 
 
 
Low Energy Availability in 
Females Questionnaire 
x 
 
 
 
 
 
 
 
Vitamin D Questionnaire 
x 
 
 
 
 
 
 
 
Blood via venipuncture 
CBC 
x 
 
 
 
 
 
 
 
TSH 
x 
 
 
 
 
 
 
 
Progesterone 
 
x 
 
 
 
 
 
 
Vitamin D (total and free 
25(OH)D) 
 
x 
 
 
 
 
 
 
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Note: The 5-day intervention, RUN or RUN+IL, occurs on days 2-6 
*pregnancy test performed at only 2 timepoints: baseline and day 1 of the 
second experimental condition after the 3-week washout period 
 
8.2 Provide a description of: 
● 
All research procedures being performed  
● 
If the study has more than one procedure, session, and/or subject 
population, describe each procedure, session, and/or study 
N-terminal propetide of type 1 
procollagen 
 
x 
 
 
 
 
 
x 
Undercarboxylated osteocalcin 
 
x 
 
 
 
 
 
x 
Sclerostin 
 
x 
 
 
 
 
 
x 
C-terminal telopeptide of type 
1 collagen 
 
x 
 
 
 
 
 
x 
Parathyroid hormone 
 
x 
 
 
 
 
 
x 
Estradiol 
 
x 
 
 
 
 
 
x 
Insulin-like growth factor-1 
 
x 
 
 
 
 
 
x 
Hepcidin 
 
x 
 
 
 
 
 
x 
Markers of iron status 
(Ferritin, iron, total iron-
binding capacity [TIBC]) 
 
x 
 
 
 
 
 
x 
Insulin 
 
x 
 
 
 
 
 
x 
Thyroid hormones (Free 
triiodothyronine (T3), free 
thyroxine and rT3) 
 
x 
 
 
 
 
 
x 
Leptin 
 
x 
 
 
 
 
 
x 
Cortisol 
 
x 
 
 
 
 
 
x 
Nesfatin-1 
 
x 
 
 
 
 
 
x 
Health outcomes 
Continuous glucose 
monitoring 
 
 
x 
x 
x 
x 
x 
 
Compliance and Fitness Testing 
Habitual dietary intake (food 
records for 3 days) 
x 
 
 
 
 
 
 
 
Habitual exercise tracking 
(smart watch for 3 days)  
x 
 
 
 
 
 
 
 
Running Economy 
 
 
x 
 
 
 
x 
 
Aerobic Capacity (VO2max) 
x 
 
 
 
 
 
 
 
Treadmill running at 65% 
VO2max 
 
 
x 
x 
x 
x 
x 
 
Physical Activity tracking 
(smart watch) 
 
 
x 
x 
x 
x 
x 
 
Mealtime Log 
 
 
x 
x 
x 
x 
x 
 
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population separately. For complex studies, you are encouraged to 
include a figure or chart. 
 
Phone Screen 
Those who respond to the investigatior’s advertisements will be scheduled to complete a 
brief telephone (or HIPAA-compliant Zoom) screening to confirm basic eligibility 
criteria.  Participants will be made fully aware of the eligibility criteria, time 
commitment, possible risks and their right to withdraw from the study at any time. A 
phone screening script and phone screening data collection form will be used for phone 
screening conducted by a registered dietitian nutritionist.  
 
Baseline Screening (approximately 1 hour) 
Informed Consent: Participants will be provided an informed consent form following the 
phone screening and in advance of coming to the laboratory.    
 
Health History:  Subjects will be asked to complete a standard health/medical history 
questionnaire, which will be used to screen for health issues (e.g., coronary or congenital 
heart disease) or other reasons (medications which influence study results) that would 
preclude participation (see uploaded Health History Screening Questionnaire). This 
questionnaire has been used by Dr. Larson-Meyer for clinical studies for the past 17 
years. 
 
Low Energy Availability in Females Questionnaire (LEAF-Q): Subjects will be asked to 
complete the LEAF-Q, which will be used to screen for risk of existing LEA, medication 
use, menstrual function, and injury history. The LEAF-Q has been validated as a 
screening tool for LEA in female endurance athletes (Melin, Tornberg et al. 2014). 
Individuals who score as “high risk” for LEA will be provided referral information to a 
local sports dietitian. 
 
Body Weight Height and Composition:  Body weight and height will be measured on a 
digital physician’s scale. Percent body fat and fat-free mass will be measured in all 
subjects via DXA scan.  
 
Bone Mineral Density: Total body, dual-femur, and lumbar spine BMD will be measured 
in all subjects via DXA scan. Scans will be conducted by T. Sterringer who is an ISCD 
Certified Bone Density Technologist. 
 
Urine sample and pregnancy test:  All participants will be required to provide a small cup 
of urine immediately before the DXA at baseline. The urine will be evaluated for 
hydration status via specific density using a refractometer. A pregnancy test will be 
performed on the urine sample for all participants.  
 
 
Baseline Laboratory Testing Session (approximately 1 hour). 
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All measurements will be performed in the morning after a 12-hour fast (no caffeine) 
with the participants instructed not to engage in heavy exercise for 36 hours prior to 
testing and to adequately hydrate the evening prior.   
 
Blood Draw: Blood will be drawn by venipuncture after a 12-hr fast (no caffeine) for the 
following: CBC, TSH, and progesterone. In total, blood will be draw five times during 
the study by venipuncture from a vein in the arm. These include baseline, and the days 
immediately before and after each experimental condition as explained below. Blood will 
be drawn with the participant seated quietly in a phlebotomy chair. Approximately 10 ml 
will be obtained during the baseline visit. 
 
Aerobic capacity (i.e., VO2max):  A graded exercise test will be performed on a treadmill 
to assess aerobic fitness via indirect calorimetry (Parvo Medics TrueOne 2400). Heart 
rate will be measured during the test by a heart rate strap and sensor. The test will begin 
with a 5-minute warm-up at 0% grade and at a speed predetermined by the participant. 
Following the warm-up phase, the workload will be increased each minute by increasing 
treadmill speed or grade by 0.5 mph or 2.5%, respectively until the participants can no 
longer continue or volitional exhaustion is reached. The entire exercise testing protocol 
will last 12-20 minutes. 
 
Habitual Diet and Exercise (3 days) 
 
Habitual Diet and Exercise: Following laboratory testing, participants will be instructed 
on completing food records for 3 days (2 weekday, 1 weekend). Food records will be 
analyzed using Nutrition Data System for Research (NDSR; University of Minnesota), a 
dietary analysis software program. Participants will also wear a smart watch over this 
same time period that will be provided by the research team. The smart watch will use an 
app to measure heart rate and estimate energy expended during structured exercise or 
physical activity; this will be used to help estimate the participants total energy 
expenditure. It will be necessary to have the GPS function turned on during the collection 
of physical activity data but only data related to time, distance or intensity and no GPS 
coordinates will be downloaded or recorded.  
 
Vitamin D questionnaire: Participants will complete a questionnaire to assess vitamin D 
status over the previous month (DE Larson-Meyer). The vitamin D questionnaire 
includes a 52-item food frequency questionnaire, 6-item supplement section, and 7 
questions related to sunlight exposure.  
 
Intervention 
After baseline screening and testing, eligible participants will be scheduled to complete 
two experimental conditions in a randomized order beginning on the second or third day 
of menstruation, depending on the participant’s schedule and available appointments. 
Each experimental condition will require participants to come into the Nutrition and 
Exercise Metabolism (NEM) laboratory on 7 consecutive days. Table 1 outlines (section 
8.1) provides an overview of data collection for each scheduled laboratory visit. On day 
1, fasted blood draws will be collected in the morning after a 12-hour overnight fast and 
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body weight will be measured on a digital physician’s scale. Participants will complete 
the LEA experimental trials on days 2-6. A follow-up blood sample will be collected on 
the morning of day 7 after an overnight fast and body weight will be measured on a 
digital physician’s scale. 
 
Pregnancy Test: a pregnancy test will be performed after the 3-week washout 
period on day 1 of the second experimental condition.  
 
Blood Draw: Blood (approximately 20-25 ml) will be drawn the days before and 
after each LEA experimental trial by venipuncture after a 12-hr fast (no caffeine) for the 
following: Progesterone, total and free 25(OH)D, N-terminal propetide of type 1 
procollagen, undercarboxylated osteocalcin, sclerostin, C-terminal telopeptide of type 1 
collagen, parathyroid hormone, estradiol, insulin-like growth factor, hepcidin, ferritin, 
TIBC, iron, cortisol, leptin, nesfatin-1, insulin, free T3, free T4, rT3. In total, blood will 
be draw five times during the study by venipuncture from a vein in the arm with a total of 
110 ml obtained for the duration of the study. Blood will be drawn with the participant 
seated quietly in a phlebotomy chair.  
 
 
Dietary Intervention: During the two experimental conditions, participants will be 
provided controlled, weighed diets of 30 kcal/kgFFM/d. Total energy of diets will be 
individualized based on the participant’s FFM measured via DXA. Diets will be prepared 
by a registered dietitian (T. Sterringer) in the research kitchen in Wallace Hall and 
standardized between conditions to include three meals and one snack. Menus will 
consist of the same whole foods and commercial products that provide diets consisting of 
approximately 55% carbohydrates, 20% protein, and 25% fat. For example, a woman 
weighing 54.5 kg (120 lbs.) with a body fat percentage of 22% (FFM = 42.5 kg) would 
be provided with a diet containing 1275 kcal (55% total calories = 175g carbohydrates, 
20% = 64g protein, 25% = 35g fat) based on 30 kcal/kgFFM (example menu uploaded). 
Participants will be instructed to consume the meals and snack at approximately the same 
time each day to avoid within-day fluctuations in energy balance (Fahrenholtz, Sjödin et 
al. 2018). They will also be instructed not to consume any other foods or beverages other 
than water and non-calorie beverages (e.g., black coffee, unsweetened tea). A daily multi-
vitamin will be provided to participants during the experimental conditions to ensure 
adequate micronutrient intake during the energy-restricted state. Participants will be 
asked to log the times they consume each meal and snack in a mealtime log for the 5-day 
experimental phases. The primary investigator (E. Larson-Meyer) is a registered dietitian 
and has extensive experience with assessment of energy balance with controlled feeding 
trials. 
 
 
Exercise Sessions: Participants will undergo supervised exercise sessions 
consisting of treadmill running with and without high-impact exercises on 5 consecutive 
days in the NEM laboratory on two occasions separated by one menstrual cycle 
(approximately 24 days). On the first day of each experimental condition, exercise energy 
expenditure (EEE) during the sessions will be measured using indirect calorimetry 
(ParvoMedics) and heart rate to ensure total EEE is approximately 15 kcal/kgFFM. In the 
RUN condition, participants will complete a 5 min warm-up (at 5.5, 6 or 6.5 mph) and 
then run on a treadmill at 65% VO2max for the duration determined at baseline (see 
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protocol in section 8.1) to elicit an EEE of 15 kcal/kgFFM. In the RUN+IL condition, 
participants will perform impact loading movements (jumping) and a running protocol. 
Participants will come to the lab in the morning to perform 5 sets of 10 impact loading 
movements for a total of 50 impacts/session at intensities greater than 2x bodyweight. 
Each jumping set will be separated by 60 seconds of rest in alignment with guidelines for 
osteoporosis prevention recommended by the Exercise and Sport Science Australia 
(ESSA) (Beck, Daly et al. 2017). Ground reaction force of impacts will be measured 
using dual force plates. Approximately 4-6 hours later, participants will return to the lab 
to complete a 5-min run and warm-up on the treadmill at 65% VO2max until a combined 
energy expenditure of the morning impact loading exercise and afternoon/evening 
treadmill running reaches 15 kcal/kgFFM. A recovery period of at least 4 hours is 
recommended between impact loading sessions for optimal osteogenic bone response 
(Hart, Nimphius et al. 2017). Participants will wear accelerometers and smart watch 
devices during the experimental conditions to measure activity level. Participants will be 
instructed to refrain from all physical activity outside of the supervised exercise sessions 
that are not related to activities of daily living (e.g., getting dressed, walking to the car).   
 
Body Weight: Body weight will be obtained before each supervised exercise 
session begins using a digital physician’s scale to monitor significant changes in body 
weight during the intervention. Participant body weight will also be needed for 
monitoring the impact of the jumping exercises using ground reaction force in the 
RUN+IL condition. The scale and monitor will be separate and daily body weight will 
not be shared with participant. 
 
Ground Reaction Force (GRF): Ground reaction force of jumping exercises will 
be performed using dual force plates. Participants will perform 5 sets of 10 vertical jumps 
with 60 seconds of rest between each set. Jumping exercises will be tailored until 
intensity reaches a threshold of at least 2x body weight of participant. These jumping 
exercises will be used in the RUN+IL condition. 
 
       Running Economy: Running economy will be assessed on the first and last day of 
each experimental condition at the start of each supervised treadmill run. Heart rate will 
be measured during the test by a heart rate strap and sensor (Polar). The test will begin 
with participant running for 4-minutes at three moderately easy speeds of 5.5, 6 6, and 
6.5 mph. The last two minutes of oxygen consumption and carbon dioxide production 
data will be used to determine metabolic economy (ml oxygen consumed per kg body 
weight per minute relative to the set work performed). This test will last 12 minutes. 
Following cumulation of the standard economy test, the workload will be increased to the 
65% VO2max. Oxygen consumption will be measured for the first 4 to 10 minutes of the 
steady state run. 
 
Continuous Glucose Monitor (CGM): Interstitial glucose concentration will be 
measured during the two, 5-day experimental conditions using a CGM device which is 
typically worn on the back of the upper arm. This requires that a small amount of 
interstitial fluid (0.5 microliters) be sampled every 15 minutes (96 times per day) 
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throughout the course of the 5-day experimental conditions. CGM will be placed on the 
day before each LEA condition and removed on the day after each condition. 
 
 
 
8.3 Describe: 
● 
Procedures or safeguards intended to reduce the probability and 
magnitude of risks. (For example: Reducing the risk of injury in a 
virtual reality study either by having the subjects sit during the study 
or by providing an obstacle-free space for walking.) 
● 
Be sure to describe all drugs and devices used in the research, when 
they will be administered or used, and their purpose.  
● 
Methods used to collect data about subjects. Please upload all data 
collection forms to Protocol Management. Some common examples 
are: 
● Screening questionnaires  
● Survey(s), including online surveys 
● Demographic questionnaire(s) 
● Interview guide(s), e.g., questions or pool of questions for semi-
structured interviews  
● Focus group guide(s) 
● Other documents used to collect data  
 
The following safeguards will be employed to reduce the probability and magnitude of 
risks associated with study participation. The specific risks are highlighted in Section 17.  
 
Energy Restriction: Potential risks associated with energy restriction include loss of lean 
body mass (LBM), suboptimal macronutrient and micronutrient intake, hypoglycemia, 
and mood disturbances. These risks will be minimized by providing a daily multivitamin 
to participants for the duration of the 5-days of energy restriction. Menus will also be 
designed to provide 20% of total calories from protein to help preserve LBM. To reduce 
risk of hypoglycemia, diets will provide 55% of total calories from carbohydrates and 
participants will be encouraged to consume the provide snack containing 30g 
carbohydrate approximately 30 minutes before the exercise sessions. Additionally, menus 
will be designed to prioritize foods with fiber and low energy density that promote 
fullness and satiety (e.g., leafy greens, fresh non-starchy vegetables, whole grains and 
other high-fiber foods) to reduce physical and psychological burden of energy restriction. 
Participants with existing menstrual disturbances (determined by progesterone and self-
report), LEA (determined by LEAF-Q score), and/or low BMD (determined by DXA) 
will be excluded from the study to prevent worsening existing conditions. 
 
High-Intensity Exercise: Potential risks associated with endurance running and high-
impact loading exercises include musculoskeletal injuries, changes in blood pressure, 
gastrointestinal discomfort, fainting, and dizziness. These risks will be minimized by 
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having participants assessed prior to each exercise session by a trained research member 
to evaluate readiness for physical activity. Additionally, participants will complete all 
exercise sessions in the lab under the supervision of a certified strength and conditioning 
specialist with CPR certification (T. Sterringer). 
 
CGM: Potential risks associated with the CGM include discomfort during the insertion, 
pain, inflammation, redness/rash, swelling, minor bleeding and minor infection at the site.  
These risks will be minimized by having a trained member of the research staff perform 
the procedure under aseptic conditions. Participants might also experience the 
aforementioned symptoms as a result of contact between the adhesive pad of the sensor 
and the skin. In rare cases, an infection can spread to other parts of the body. Allergic 
reactions can develop in response the adhesive used to keep the CGM in place. If these 
symptoms occur, participants have the ability to remove the CGM at will. Symptoms 
typically resolve within a short time (approximately one week). 
 
Questionnaires and Study Logs: All study questionnaires (except the food records) will 
be collected with the participant sitting in a private setting in the laboratory. 
Questionnaires will be placed in each participant’s study file date entered for data 
analysis. 
 
Blood draws: Blood will be collected using universal precautions by a trained technician. 
Blood will be drawn by venipuncture from a vein in the arm with the participant resting 
in a phlebotomy chair.  Blood will be drawn at five times during the study (baseline, 
RUN day 1, RUN day 7, RUN+IL day 1, RUN+IL day 7) with a total of 110 ml of blood 
collected over the course of the 5-6 weeks. 
 
DXA scan: Participants will be exposed to a very low dose of ionizing radiation as part of 
the DXA scan at baseline only. DXA procedure will be performed by trained staff. 
Participants will be informed of the risk of radiation exposure prior to study enrollment. 
Female participants will complete a pregnancy test by urine immediately before the 
DXA. 
 
VO2max/Aerobic capacity: Trained research personnel will be present during the test to 
correctly place the mouthpiece, monitor all variables during the test and support the 
participant at the end of the test.  
 
8.4 What data will you collect during the study and how you will obtain them? 
Please include descriptions of electronic data collection, database 
matching, and app-based data collection: 
 
Anthropometric and basic demographic (age) data will be recorded on data sheets and 
manually entered into a database (excel format) on a secure computer. Select data (DXA 
results, VO2max, ground reaction force) may be transferred electronically directly from 
the DXA, metabolic cart, or force plates into excel spread sheets, if possible. Blood 
results will be entered directly from laboratory sheets provided by a commercial 
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laboratory or the Metabolic Core at Virginia Tech. Heart rate, energy expended and 
exercises performed will be downloaded onto the laboratory computer from the Smart 
Watch. Glucose concentration in interstitial fluid samples by time will be downloaded 
directly from the CGM sensor onto the lab computer.   
 
8.5 Who will transcribe or code audio and/or video recordings?: 
 
N/A 
 
8.6 Include a description of any deception to be used in the study. Include 
justification for the use of deception (why the deception is necessary), 
describe the debriefing process, and describe how the study meets all the 
following criteria  for alteration of consent (deception is considered an 
alteration of informed consent): 
• The research involves no more than minimal risk to the subjects 
• The alteration will not adversely affect the rights and welfare of the 
subjects 
• The research could not practicably be carried out without the 
alteration/deception 
• (Optional but encouraged in most cases) Subjects will be provided with 
additional pertinent information after participation (i.e., debriefing for 
studies involving deception)  
 
 
N/A 
 
 
8.7 If the study involves long-term follow-up (once all research related 
procedures are complete), describe what data will be collected during the 
follow up period and when it will occur:  
 
N/A 
 
9.0 
Data and Specimen Long Term Storage and Use 
9.1 If you will store data or specimens for future use, describe where you will 
store the data or specimens, how long they will be stored, and how and by 
whom the data or specimens will be accessed: 
 
All data will be stored in a locked cabinet in Dr. Larson-Meyer's laboratory which will 
also be locked to only authorized personnel. The computer data will be stored in the 
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locked lab on a computer that is password protected. All de-identified data will be kept 
indefinitely.  
 
9.2 For specimens, list the data to be stored or associated with each 
specimen: 
Blood and urine samples will be stored in a -80-degree freezer in the HIP 
laboratory currently located in the Garvin Building. Samples will be 
labeled with the participants’ study code (see section 9.4 below), the visit 
number and the date and time of the collection. No identifying information 
will be written on specimen samples. The freezer is located in locked 
room/laboratory. 
 Blood analyzed by the Metabolic Core at Virginia Tech, housed in the 
Integrated Life Sciences Building, may also be temporarily stored in a 
freezer in this laboratory immediately before, during or after analysis.   
 
9.3 Describe the procedures to release data or specimens outside of the 
research team, including the process to request a release, approvals 
required for release, who can obtain data or specimens, and what data 
will be provided with specimens: 
 
Some de-identified blood and urine samples will be sent to a commercial 
laboratory for analysis based on cost savings.  There are currently no plans 
to release data outside of the research team.  
 
9.4 Describe the identifiers to be included with stored data or specimens, as 
well as any key or code that could be used to make them identifiable. 
Describe where the code will be stored, who will have access to it, and 
when it will be destroyed: 
 
Study Codes using a combination of letters and numbers will be used to de-identify 
subjects from their personal information. No obvious identifiers will be stored with the 
data; the data spreadsheet, however will include each participants’ age and starting 
weight as part of the de-identified data.  Original de-identified data collection sheets will 
be stored in a locked file cabinet as part of study records; scans of some de-identified 
information may be kept in a password-protected electronic file that is accessible only to 
research personnel. During the active phase of the study, a master document (key) that 
will contain the participants name, assigned study code and randomization order will be 
kept in a password-secured file that will be accessible only to the PI and authorized study 
personnel (doctoral student in charge of the study, T. Sterringer). The key will be 
destroyed 6 to 12 months after collection of data from the last participant. De-identified 
data may be kept indefinitely. Blood and urine samples will be destroyed after 5 years. 
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9.5 Please select the identifiers you will obtain (whether directly from 
participants or from another source), including but not limited to: 
 
☒ 
Name 
☒ 
Geographical subdivisions smaller than a state, including street address, 
city, county, precinct, zip code, and equivalent geocodes (note, the initial 
three digits of a zip code are not considered identifiable) 
☒ 
Elements of dates (except year) for dates directly related to an individual, 
including birth date, admission date, discharge date, date of death, and 
single year of age over 89 and all elements of dates (including year) 
indicative of such age (note, such ages and elements may be aggregated 
into a single category of age 90+) 
☒ 
Phone numbers 
☐ 
Fax numbers 
☒ 
Electronic mail addresses (e-mail) 
☐ 
Social Security numbers 
☐ 
Medical record numbers 
☐ 
Health plan beneficiary numbers 
☐ 
Account numbers 
☐ 
Certificate/license numbers 
☐ 
Vehicle identifiers and serial numbers, including license plate numbers 
☐ 
Device identifiers and serial numbers 
☐ 
Web Universal Resource Locators (URLs) 
☐ 
Internet protocol (IP) address numbers 
☐ 
Biometric identifiers, including finger and voice prints (audio recording) 
☐ 
Full face photographic images and any comparable images (including 
video recording) 
☐ 
Student record number or identification number   
☐ 
User name for online or computer accounts 
☐ 
Any other unique identifying number, characteristic, or code (note this 
does not mean the unique code assigned by the investigator to code the 
data): Click here to explain.   
 
10.0 Sharing of Results with Subjects 
10.1 Describe whether you will share results (study results or individual subject 
results, such as results of investigational diagnostic tests, genetic tests, or 
incidental findings) with subjects or others (e.g., the subject’s primary care 
physician). If so, describe how you will share the results and include this 
information as part of the consent document. Upload materials you will use 
to explain the results to subjects: 
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At the conclusion of the study or when the participants' involvement in the study ends, 
interested participants will be provided with individual results related to their body 
composition, BMD, fitness, and pertinent blood markers. These data will be summarized 
on a TBD summary document that the participant can pick up at the lab or have (upon 
request) mailed to them at a provided address. This form will be submitted as an 
Addendum before it is provided to the first participant. Participants will only be provided 
results during the study if it is determined that the participant has a result for any 
measured outcomes that is out of the normal range; in this case the participant would be 
provided information about the value and asked to see their personal health care provider. 
The participants will also be notified when a summary of the study findings is published 
if an active email address is on file.   
 
11.0 Study Timelines 
11.1 Describe: 
● 
The duration of an individual subject’s participation in the study (for 
example, 1 hour, 2-4 weeks, 3-5 years). 
● 
The amount of time expected to enroll all study subjects (weeks, 
months, years, etc.) 
● 
The amount of time expected for the investigators to complete this 
study including primary data analyses. 
The duration of an individual's participation in this study will be approximately 6 to 10 
weeks, which will include a Zoom or telephone call for screening, a screening visit in the 
laboratory, a baseline visit, three days of habitual diet and exercise tracking, two 
experimental conditions, and a washout period of 1 menstrual cycle (approximately 3 
weeks). The actual time and frequency of the subject's visits will depend on their 
schedule and that of the study staff. Participants will begin the study on a rolling basis, 
but the entire study will take place across approximately 6 months based on enrollment of 
1-2 participants per week. The investigators will complete primary data analyses within 
the following year but all analyses of study data may not occur for up to ten years 
following study completion.  
 
12.0 Inclusion and Exclusion Criteria 
12.1 Describe how you will screen individuals for eligibility. When will 
screening occur and what procedures will you use? Upload any screening 
scripts or surveys to Protocol Management:  
Those who respond to the investigation's advertisements will be asked to complete a brief 
telephone (or Zoom) screening to confirm basic eligibility criteria. Participants will be 
made fully aware of the eligibility criteria, time commitment, possible risks and their 
right to withdraw from the study at any time. A phone screening form will be used for 
this purpose conducted by a research team member who is also a registered dietitian 
nutritionist (RDN). The Low Energy Availability in Females Questionnaire will be used 
to screen for risk of existing LEA, medication use, menstrual function, and injury history. 
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12.2 Describe the eligibility criteria that define who will be included and who 
will be excluded from enrollment for each procedure of your study. 
Include any geographic criteria (e.g., Virginia Tech undergraduate 
students, a national sample of adults with engineering degrees, minors 
aged 8-12 in the New River Valley, university faculty in Virginia and 
Paris, France): 
 
Eligible participants will be well-trained, eumenorrheic female runners between the ages 
of 18-35 years that are weight-stable with a BMI between 18.5-30 kg/m2. Participants 
with LEAF-Q scores ≥8, existing menstrual disturbances measured by progesterone and 
self-report, or low BMD (z-score <-2) will be excluded from the study to prevent 
worsening existing conditions and recommended to follow up with their primary care 
physician and/or a registered dietitian nutritionist. During the phone screening, 
participants will be asked if they have ever been diagnosed with an eating disorder and 
the circumstances surrounding the diagnosis. If the participant has just recently recovered 
(within the last 12 months) or is still in recovery, she will be excluded from the study. 
The LEAF-Q was not developed to assess disordered eating behavior, however, a study 
in ultra-marathon female runners found a significant association between high LEAF-Q 
scores and disordered eating (Folscher, Grant et al. 2015). Athletes who score 8 or higher 
on the LEAF-Q will be excluded from the study and referred to a local sports dietitian. 
Participants using contraceptives (oral contraceptives, injections, IUD, etc.) will be 
excluded from this study. Contraceptive use may mask menstrual irregularities and oral 
contraceptives may influence glucose metabolism (Lopez, Schultz 2007), a secondary 
outcome of this study. Adequate training status for the study protocol will be assessed 
based on running volume, frequency, and VO2max. Eligible participants must be able to 
run on 5 days/week for at least 60 minutes to meet the training requirements of the study 
protocol. Additional exclusion criteria include history of fracture in previous 6 months,  
medication use that could potentially affect bone metabolism (e.g., corticosteroids, 
anticonvulsants, heparin, gonadotropin-releasing hormone agonists), pregnancy, 
lactation, abnormal TSH, and routine engagement in mechanical loading exercises. 
Participants must be willing to consume the diets provided, however, diets will be 
modified based on patient allergies or preferences, within reason. Participants will be 
excluded if they have dietary restrictions or preferences that would prevent them from 
consuming meals that fit within the experimental conditions. For example, we cannot 
modify diets to meet low-carbohydrate preferences given the experimental diets must 
contain 55% of total calories from carbohydrates. Similarly, participants who are unable 
to consume high-fiber diets will be excluded. 
 
12.3 Indicate specifically whether you will include or exclude each of the 
following special populations: (You may not include members of these 
populations as subjects in your research unless you indicate them in the 
description of your subject population.) 
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● 
Minors, as defined by state law where the study is performed 
(infants, children, teenagers) 
● 
Pregnant women (can be included in minimal risk studies by 
mentioning in section 13.1) 
● 
Prisoners (including all incarcerated individuals) 
● 
Adults not capable to consent on their own behalf 
 
None of the above will participate. 
Pregnant women are excluded because intentional energy restriction is not appropriate 
during pregnancy.  A pregnancy test will be performed at baseline prior to the DXA scan 
and after the 3-week washout period on Day 1 of the second experimental condition. 
 
13.0 Vulnerable Populations 
13.1 If the research involves individuals who are vulnerable to coercion or 
undue influence, please describe additional safeguards you will include to 
protect their rights and welfare. Consider the applicable items listed 
below: 
● 
If the research involves Virginia Tech students, indicate whether 
these are students of any of the investigators. If so, describe whether 
the activities will take place during class time as part of the 
curriculum and the steps you will take to reduce the possibility that 
students feel obliged to participate in order to improve their course 
grade. The HRPP can provide further guidance as needed. Describe 
whether you will request access to student records (e.g., SAT, GPA, 
GRE scores). 
● 
If the research involves employees of Virginia Tech or the research 
sponsor, describe steps you will take to ensure that the employees 
are freely participating and describe how their data will be 
protected from inspection by their supervisors.  
● 
If the research involves Virginia Tech NCAA athletes, you must 
obtain approval from the athletic department.  
● 
For research involving Montgomery County Public Schools, you 
must obtain county approval (after obtaining contingent Virginia 
Tech approval). Other locales have different requirements; please 
check on these and describe here. Approval is typically granted by 
the superintendent, principal, and classroom teacher (in that order). 
Approval by an individual teacher is insufficient. School approval, in 
the form of a letter or a memorandum should be uploaded as a 
supporting document. 
● 
If the research involves pregnant women, review “CHECKLIST: 
Pregnant Women (HRP-412)” to ensure that you have provided 
sufficient information in this protocol. 
● 
If the research involves prisoners, review “CHECKLIST: Prisoners 
(HRP-415)” to ensure that you have provided sufficient information 
in this protocol. 
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● 
If the research involves persons who have not attained the legal age 
for consent to treatments or procedures involved in the research 
(minors), review the “CHECKLIST: Minors (HRP-416)” to ensure 
that you have provided sufficient information in this protocol. 
● 
If the research involves cognitively impaired adults, review 
“CHECKLIST: Cognitively Impaired Adults (HRP-417)” to ensure 
that you have provided sufficient information in this protocol. 
 
This research study has the potential to include students and employees of Virginia Tech. 
However, during the consenting process, the participants will be made aware that only 
members of the research study team will have access to their data and that this data will 
utilize a coding system making their data unidentifiable. This data will be locked away 
and they will be made fully aware of their right to withdraw from the study at any time.  
If Virginia Tech athletes are interested in the study, they will only be allowed to 
participate during their off-season and after first obtaining approval from the athletics 
department. 
 
 
 
14.0 Number of Subjects 
14.1 Indicate the total number of subjects to be enrolled and how this number 
was determined (e.g., sample size calculation [show], number of available 
subjects in a finite pool, number of tests funding award would allow):  
 
A power analysis determined that 12 women will be required to detect a 
clinically significant change of 10μg/L in PINP with 80% power at P < 
0.05 based on results from a similar study of LEA during dietary 
restriction (Papageorgiou, Martin et al. 2018). We aim to enroll 20 
participants to account for possible dropout. 
 
14.2 If this is a multi-site study, indicate the number of subjects to be enrolled 
at this site and the total to be enrolled from all sites: 
 
N/A 
 
14.3 If applicable, indicate the number of potential subjects you expect to 
screen for enrollment, and the number of subjects you will need to 
complete the research procedures: 
 
We anticipate that we may need to screen between 50-60 participants to 
recruit and complete the required 12 participants. 
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14.4 If the study has more than one procedure, indicate the total number of 
subjects to undergo each procedure separately: 
 
All enrolled participants will undergo the two experimental procedures.  
 
15.0 Recruitment Methods 
15.1 Describe when, where, and how you will recruit potential subjects: 
 
Participants will be recruited through flyers placed at strategic locations 
(gyms, fitness, and recreational centers, etc.) at colleges and universities in 
the New River Valley (including Virginia Tech), local running stores, 
running groups in New River Valley, and through targeted listservs (VT 
News), emails, and social media posts.  
 
15.2 Describe the source of subjects (for example, clinic patients with specific 
conditions, students in the library, community members at a gathering, or 
members of a local gym): 
 
We will recruit from the general population of athletes and active 
individuals. This will include recruiting members of local running groups 
and residents of New River Valley. We welcome diverse participants from 
all racial, ethnic, educational, financial, and social backgrounds. Given the 
nature of this laboratory-based intervention, subject recruitment will be 
limited to individuals residing in the New River Valley and surrounding 
areas. Increased recruiting efforts will be targeted at Radford City County 
given the increased diversity of this county compared to the other 4 
counties in New River Valley. 
 
15.3 Describe the methods that you will use to identify potential subjects: 
 
As mentioned previously above (15.2), we will identify participants 
through use of flyers placed at strategic locations and through targeted 
listservs, emails and social media posts.   
 
15.4 Describe materials that you will be use to recruit subjects. Attach copies 
of these documents with this protocol in Protocol Management and be 
sure to include the IRB protocol number on each document.  
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● For flyers, attach the final copy of printed flyers.  
● For Virginia Tech News, Facebook postings and ads, newspaper ads, 
websites, MTurk/SONA/online survey systems, etc., attach the final 
wording and graphics to be used.  
● For email recruitments, please include the subject line.  
● For advertisements meant for audio broadcast, please submit the 
wording of the advertisement prior to taping (to avoid having to re-
record with approved language) and submit the final recorded version 
for IRB review before use.  
● Describe any compensation to subjects. Separate compensation into 
appropriate categories, such as: reimbursement for expenses, time and 
effort, and additional incentives for study participation.  For each 
category, specify the amount (including any pro-rated amount), schedule, 
and method of payment. 
 
A draft copy of our recruitment flyer is uploaded.  This flyer will be posted at 
strategic locations throughout Blacksburg and the surrounding area. We will seek 
permission at each site as necessary before posting or hanging a flyer. We also 
plan to use this same advertisement for emails and a modified version for social 
media posts (that will be submitted for approval at a later date as an Addendum).  
Emails will use the subject line "Volunteers needed for study on bone health of 
long-distance runners". Participants will be compensated $25 for completing the 
baseline visit and first experimental condition (7 days). Participants will be 
compensated an additional $75 for completing the second experimental 
condition. This is a total of $100 compensation for completing all testing visits 
and experimental conditions. Participants that do not complete the second 
condition will receive information about body composition, BMD, and aerobic 
fitness (VO2max, running economy), in addition to the $25 compensation. Given 
the cross-over design of this study, data cannot be used for participants that do 
not complete both conditions. The experimental conditions will be separated by 3 
weeks of their normal diet and exercise routine and not require any lab visits or 
intervention. The total time commitment for this study will be 19 days. Payment 
in the form of cash will be scheduled after each individual participant completes 
each experimental condition.  
 
 
16.0 Withdrawal of Subjects 
16.1 Describe circumstances under which you anticipate subjects could be 
withdrawn from the research without their consent: 
 
Participants could be withdrawn from the study if they are not showing up 
for appointments and/or exercise sessions, are not consuming the provided 
diets, or are not completing or complying with all procedures. They also 
may be withdrawn if they develop and injury or illness that would prevent 
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them from doing everything that is expected for the study or which might 
compromise their health.  
 
16.2 If applicable, describe any procedures for orderly termination (e.g., 
discontinuation of a study drug or debriefing after a behavioral 
intervention): 
 
If a participant is not complying with the study, the PI or another member of the study 
staff will first discuss these difficulties with the participant and explain the importance of 
adhering to the intervention for the purpose of the study. If it is determined that the 
participant be terminated or discontinued from the study for reasons as described above, 
the PI will mitigate issues leading to these problems. The participant will be provided any 
information which is available to them (baseline body composition, fitness testing, 
BMD). It will then be suggested that the study personnel part ways with the participant. 
 
16.3 Describe procedures that you will follow when subjects withdraw from the 
research, including partial withdrawal from procedures with continued 
data collection (e.g., participant declines to continue with regular blood 
draws, but continues with periodic behavioral questionnaires): 
Any participant can discontinue participation at any point without consequence. 
 
17.0 Risks to Subjects 
17.1 List the reasonably foreseeable risks, discomforts, hazards, or 
inconveniences to the subjects related the subjects’ participation in the 
research. Include for the IRB’s consideration a description of the 
probability, magnitude, duration, and reversibility of the risks. Consider 
physical, psychological, social, legal, privacy, and economic risks. Do not 
indicate “No risk” or “N/A.” Instead, for studies with very low risk (e.g., 
anonymous online questionnaire on a mundane topic) indicate “The 
investigators are not aware of any risks from participation in this study.” 
or “No more than risks than are found in everyday life.” The example 
consent form presents a tabular method for risk information, which you 
can also use here. Common risk types include: 
● Physical (e.g., potential for pain, discomfort, infection) 
● Psychological (e.g., potential for stress, discomfort, and/or 
embarrassment) 
● Social (e.g., potential for discrimination or stigmatization and 
disruption of personal and family relationships) 
● Legal (e.g., potential for disclosure of illegal activity, negligence) 
● Privacy (e.g., potential for personal information being accessed, used, 
or disclosed without the subjects’ knowledge or consent, breach of 
confidentiality/security) 
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● Economic (e.g., potential for individuals to lose access to economic 
services, employment, insurability) 
 
DXA Scan: The amount of radiation that subjects will receive in the DXA exam is less 
than the amount permitted by the Food and Drug Administration (FDA) per year. The 
amount subjects will receive at the exam (including total body, femur, and lumbar spine) 
is equal to 1/20 of a chest x-ray. The more radiation an individual receives over the 
course of their lifetime, the more likely that individual’s risk increases in developing 
cancerous tumors. The radiation in this study is not expected to greatly increase these 
risks, however, the exact increase in such risk is not known. 
 
Blood draws: Slight discomfort may be expected during blood draws. Risk of developing 
a small bruise or blood clot in the vein, risk of fainting or dizziness, risk of infection and 
risk of bleeding are also possible. Universal Precautions will be followed for collection, 
handling, processing, and disposal of items that may have come into contact with bodily 
fluids during the collection of blood. Blood draws will be performed by a research 
phlebotomist (J Rinehart) trained and experienced in the blood draw procedure and in 
handling minor emergencies such as dizziness and fainting. In case of an emergency, 911 
will be called. 
 
Running Economy, Exercise Capacity (VO2max), and Treadmill Runs:  There is a small 
risk of orthopedic injury, treadmill falls or cardiovascular complications that could 
require a participant to go to the hospital. This includes a heart attack, or even death.  In 
studies involving people with heart disease, the risk of hospitalization was 1 in 500 tests 
(<0.20%). The risk of heart attack was 1 in 2,500 tests (0.04%) and death, 1 in 10,000 
tests (0.01%). The risks are likely to be lower in young, healthy participants who are 
involved in running and other exercise activities.  Only experienced staff members will 
conduct these tests and subjects will be monitored throughout the test for signs of 
problems based on standards of the American College of Sports Medicine (ACSM).  
There is a possibility some subjects will be tired after this test and could have sore 
muscles for a few days. Risks associated with treadmill running will be minimized by 
recruiting only participants who have current experience with long-distance running at a 
high volume (at least 30 miles per week) and frequency (at least 5 days per week) to meet 
the exercise demands of the protocol. 
 
Continuous Glucose Monitoring. The placement of the device will require that a sensor is  
inserted into the back of the participant’s upper arm. Placement of the sensor may  
induce some pain during the insertion, inflammation, redness, swelling, 
minor bleeding and/or minor infection at the site. This will all be minimized by having a 
trained individual perform the procedure which will take place in aseptic conditions. 
There is also a possibility a participant may experience these symptoms as a result of 
contact between the adhesive pad of the sensor and the skin; allergic reactions can also 
develop in response to the adhesive used to keep the device in place. 
If any of these symptoms occur, the participant will be informed that he/she has the 
ability to remove the CGM and these issues will clear up within a short time period. 
 
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Dietary Intervention: Experimental diets will induce a state of low energy availability 
through dietary energy restriction. Potential risks associated with energy restriction 
include loss of lean body mass (LBM), suboptimal macronutrient and micronutrient 
intake, hypoglycemia, feelings of weakness and mood disturbances. These risks will be 
minimized by providing a daily multivitamin to participants for the duration of the 5-days 
of energy restriction. Menus will also be designed to provide 20% of total calories from 
protein to help preserve LBM. To minimize risk of hypoglycemia, diets will provide 55% 
of total calories from carbohydrates and a snack will be provided to participants 
containing approximately 30g carbohydrate to be consumed prior to the treadmill run. 
Additionally, menus will be designed to prioritize foods with low energy density that 
promote satiety (e.g., high fiber foods) to reduce physical and psychological burden of 
energy restriction. Participants with existing menstrual disturbances, LEA, and/or low 
BMD will be excluded from the study to prevent worsening existing conditions. Given 
the short-duration of this study and the washout period of approximately 24 days between 
LEA conditions, it is unlikely the acute energy restriction will result in any long-term 
consequences. 
 
High-Impact Loading Exercises (Jumping): Potential risks associated with high-impact 
loading exercises include musculoskeletal injuries and excessive bone strain. These risks 
will be minimized by excluding participants with recent history of bone stress injuries or 
low BMD (z-score <-2.0). Additionally, the exercise sessions will be supervised by an 
NSCA-certified strength and conditioning specialist (T. Sterringer) with an emphasis on 
proper form and safety techniques. The participants will be informed to contact the PI or 
any member of the research team in the case of any injury or excessive joint or muscle 
strain.   
 
17.2  Indicate the measures you will use to minimize risks and monitor subjects 
for safety. (e.g., asking a subject at regular intervals to rate how they are 
feeling from 1 to 10, or to slowly crouch in order to check their balance.) 
Indicate the measures you will use to minimize risks and monitor subjects 
for safety. (e.g., asking a subject at regular intervals to rate how they are 
feeling from 1 to 10, or to slowly crouch in order to check their balance.) 
 
Weight loss will be expected during the two, 5-day intervention periods because 
the participants will be in a state of low EA (15 kcal/kgFFM/d). Previous studies 
(Ihle, Loucks 2004, and Loucks, Thuma 2003) observed an average weight 
reduction of 2.0-2.2 kg and 1.1-1.2 kg in young women (average age 21 years) in 
response to EA treatments of 10 and 20 kcal/kgFFM/d, respectively, over a 5-day 
period. This study will use a safety endpoint of excessive weight loss defined as 
5-lbs (approximately 2.3 kg) over the 5-day energy-restricted intervention 
phases. 
 
Other safety measures addressed in Section 17.1 
 
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17.3 If applicable, indicate which procedures might have risks to the subjects 
that are currently unforeseeable. This will be rare, and usually applicable 
when testing a new drug or device or a new use of an existing drug or 
device: 
 
It is possible that participants could develop soreness in legs by 
participating in our high-volume running regimen on 5 consecutive days 
and during the 5-day intervention with high-impact jumping. It is also 
possible an unknown allergy to foods contained in the controlled diets 
could be identified during the study. These events, however, are not likely. 
To minimize the potential, we will only recruit participants with current 
long-distance running experience at a high volume (at least 30 miles per 
week) and frequency (at least 5 days per week) to meet the exercise 
demands of the protocol.  
 
17.4 If applicable, indicate which procedures might have risks to an embryo or 
fetus should the subject be or become pregnant: 
 
Dietary restriction is a risk to the development of the fetus should the subject become 
pregnant during the study intervention. Pregnancy tests will be performed at baseline 
testing (specifically before the DXA) and after the 3-week washout period on Day 1 of 
the second experimental condition, to ensure the participant is not pregnant. Progesterone 
will be measured to ensure menstrual status. Participation in the study would end if it 
were determined the participant has become pregnant.  
 
 
17.5 If applicable, describe risks to others who are not subjects (e.g., collection 
of sensitive health data that might affect sexual partners if disclosed, 
mandatory reporting of abuse, DNA testing that might affect family 
members or relationships): 
 
N/A 
 
18.0 Potential Benefits to Subjects 
18.1 Describe the potential benefits that individual subjects might experience 
from participating in the research. Include the probability, magnitude, and 
duration of the potential benefits, as this will be useful to the IRB’s 
risk:benefit analysis. Do not include benefits to society or others. Do not 
list monetary or non-monetary compensation for participation, as this is 
not a benefit These should be included in section 2 or 3 of this document: 
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Participants will gain information about their bone mineral density, body 
composition and running fitness (VO2max and running economy). They 
will also learn how to properly perform high-impact loading exercises.  
 
18.2 If applicable, specify that there are no anticipated direct benefits for 
participants:  
 
N/A 
 
 
19.0 Data Management and Confidentiality 
19.1 Describe procedures that you will use for quality control to ensure validity 
of collected data: 
 
Dr. Larson-Meyer has extensive experience performing data collection the 
procedures (or similar procedures) as does Dr. Elaina Marinik. Dr. 
Marinik and Trisha Sterringer are ISCD Certified Bone Densitometry 
Technologists. Additionally, T. Sterringer is a NSCA Certified Strength 
and Conditioning Specialist and has experience or been trained on 
performing the procedures detailed in this protocol. The research team will 
ensure all study personnel will be properly trained to perform all 
procedures according to standard protocol. Specific quality control 
measures will be employed to ensure valid indirect calorimetry data are 
collected during the exercise economy, VO2max, and exercise energy 
expenditure tests; These standards are included on data collection sheets 
for use by members of the study team.  
 
19.2 Describe any existing data or biospecimens you will obtain as part of this 
study. Include:  
● 
Variables or samples to be obtained  
● 
Source of the data or specimens 
● 
Your authorization to access or receive the data or biospecimens 
● 
Whether the data or biospecimens are publicly available 
● 
Whether the data or specimens you receive will contain identifiers  
 
N/A 
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19.3 Describe the steps that you will take to handle and secure study data 
during data collection, storage, use, and transmission.  Include 
information about training of study staff, authorization of access, 
password protection, encryption, physical controls, certificates of 
confidentiality, separation of identifiers and data, etc.: 
 
We will do everything that we can to make sure that study records are kept private. Each 
participant will be assigned a unique participant code as explained in section 9.4.  All 
data recording sheets and spread sheets will use the subjects’ study code. They will not 
contain the participants’ name or date of birth. These will be compiled in a patient 
research file/chart and stored in a locked file cabinet organized by their unique study 
code. Their name will be listed only on the phone screening log (which is to be blackened 
out after they are assigned a participant code), informed consent and on a master 
participant list that includes the randomization key. The master participant list will be 
kept in a separate electronic file than the data files; both will be password-protected. The 
study consents will be kept together in a separate file in a separate location in a locked 
office. Only authorized study personnel will have access to study data.  Results of the 
study may be published and/or presented at professional conferences. The participants’ 
name or other personal information that would identify them will not be used. All blood 
collected and post-processed serum and plasma samples will be labeled with the 
participants unique study code (plus the study visit and date and time of sample 
collection) and stored in a secure freezer in a locked laboratory until analysis as 
mentioned in section 9.2. Archives may be kept for up to five years following study 
analysis. Training of study personnel including graduate students on procedures to ensure 
secure collection and storage of study data will occur before study initiation.  
 
19.4 For multi-site studies, describe how data or specimens will be handled 
and secured for each site (e.g., central or disseminated data storage, data 
coordinating center): 
 
N/A 
 
19.5 Describe the plan for data disposition following the conclusion of the 
study (e.g., long term maintenance of data, data destruction methods).  
● 
What information will be included in the long term storage of data 
or specimens? 
● 
How long will the data or specimens be stored? 
● 
Where and how data or specimens will be stored? 
● 
Who will have access to the data or specimens during long term 
storage? 
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● 
Who is responsible for receipt or transmission of the data or 
specimens? 
● 
How will data or specimens be shared or transported? 
● 
When and how will personal identifiers be destroyed? 
 
 
Telephone screening forms (that contain participants’ names) will be shredded 
immediately after all study participants are recruited. Personal information, 
primary and secondary endpoints and safety data will be kept indefinitely in a 
secured electronic location by the PI. Personal information will be kept in a 
separate file than de-identified data. Blood and urine samples labeled with the 
patients' unique study code may be stored in a laboratory freezer in a locked 
laboratory for up to five years following the completion of the analyses; only 
authorized study personnel will have access to freezer samples. The PI will be 
responsible for transmission of all data or achieved specimens.  Although is not 
anticipated that any data will need to be transported or shared, this would be 
done only using de-identified data with samples sent using a secure mechanism.  
 
20.0 Provisions to Protect the Privacy Interests of Subjects 
20.1 Describe the steps that you will take to protect subjects’ privacy interests. 
“Privacy interest” refers to a person’s desire to place limits on with whom 
they interact or to whom they provide personal information (e.g., 
collecting the minimal amount of private information required to complete 
the study, protecting the data once it is obtained):  
 
To ensure privacy interests of all interested and enrolled participants, only 
the minimal amount of personal information and health history will be 
obtained using a standard health history form and the Low Energy 
Availability in Females Questionnaire. This data will be kept in participant 
files labeled with only the participants’ study code in a secured file in a 
locked room. The data for all participants who do not participate in the 
study will be destroyed by shredding. The data for participants who do 
enroll will be entered into an electronic data base using the participants 
assigned unique study code. Any and all original data collection sheets 
with the participants’ name or identifying information will also be 
destroyed following entry into the database.   
 
20.2 Describe steps that you will take to make subjects feel at ease with the 
research situation in terms of the questions being asked and the 
procedures being performed. “At ease” does not refer to physical 
discomfort, but the sense of intrusiveness a subject might experience in 
response to questions, examinations, and procedures (e.g., use of a same 
gender investigator to place sensors on the torso, a private changing area 
if clothing must be changed, sensitivity when discussing  pregnancy testing 
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with subjects, making it clear on surveys that participants can discontinue 
at any time, not asking questions about private or sensitive issues unless 
necessary for the research): 
 
Study participants will be informed during the baseline screening and 
exercise sessions that they can discontinue the study and/or exercise 
session at any time without penalty. All questionnaires and anthropometric 
testing will be performed by trained research personnel in a private setting. 
Assignment of same-sex researchers will be employed if necessary; 
however, all study personnel will be trained to exhibit professional 
behavior and sensitivity when collecting personal health or medical data or 
when performing body composition or other testing.  
 
20.3 Describe how you plan to access existing sources of information about the 
subjects (e.g., medical records, grades) and how you will protect 
participant privacy through the data security plan: 
 
N/A 
 
20.4 Describe any required reporting that might occur as a result of your 
research questions, study populations, and data collection methods. 
Examples for Virginia and Virginia Tech include: 
• Any suspicions (e.g., circumstantial, disclosed) of child abuse (physical, 
emotional, sexual) and neglect  
• Sexual discrimination and/or sexual violence that involves a student 
• Disclosure or signs of intention to harm oneself (i.e., suicidal ideation 
and/or plan) 
• Disclosure or signs of desire to harm others (i.e., homicidal ideation 
and/or plan) 
• Suspected abuse, neglect or exploitation of vulnerable adults (e.g., 
individuals with a disability, elderly persons) 
 
 
N/A 
 
 
 
21.0 Provisions to Monitor the Data to Ensure the Safety of Subjects 
Safety monitoring is required when research involves greater than minimal risk 
and is sometimes appropriate for other studies. 
21.1 Describe: 
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● 
The plan to periodically evaluate the data collected regarding both 
harms and benefits to determine whether subjects remain safe  (e.g., 
periodic reporting to the IRB, establishing a data monitoring 
committee, reporting data monitoring committee findings to the IRB 
and the sponsor). 
● 
What data you will review, including safety data, unexpected events, 
and data that show the ability to produce the intended results. 
● 
How the safety information will be collected (e.g., with case report 
forms, at study visits, by telephone calls with subjects). 
● 
The frequency of data collection, including when safety data 
collection starts. 
● 
Who will review the safety data and with what frequency. 
● 
The statistical tests for analyzing the safety data to determine 
whether harm is occurring. 
● 
Any conditions that will trigger an immediate suspension of the 
research (e.g., a serious adverse event). 
 
 
 
The data safety monitoring plan (DSMP) for this study focuses on close monitoring by 
the principal investigator (PI) and research staff along with prompt reporting of excessive 
adverse events and any serious adverse events (AEs) to the Institutional Review Board. 
All serious AEs will be reported by the PI within 48 hours of occurrence to the IRB and 
the sponsor. 
  
The safety data monitored will include data related to the blood collections, fitness testing 
and the supervised exercise sessions. Specific safety data include any reports of pain, 
excess swelling, redness or bruising after the blood draws at the needle insertion site, 
feelings of light headedness, chest tightness or pain or fatigue on exertion during exercise 
testing procedures, and symptoms of muscle soreness, joint pain, or unexpected 
events/issues during the 5 days of consecutive running and 5 days of consecutive running 
with impact loading exercises. Data will be collected and documented in the participant’s 
chart if a situation arises or when observed by a member of the research team or reported 
by a participant during a study visits or during supervised resistance training sessions 
using a general TBD incident reporting form. Safety data will also include excessive 
changes in body weight or interstitial glucose concentration that will measured 
throughout the 5 days of energy restriction in the experimental conditions. 
 
The graduate student in charge of the project (T. Sterringer) will consult with Drs. 
Larson-Meyer and Marinik and be responsible for assembling the data, producing reports, 
and assuring that all parties obtain copies of these reports. Reports will be submitted 
annually to the VT IRB for review. 
  
Safety Data collection will start when the first participant is screened and enrolled. The 
study team will be informed to discuss any observed or reported unusual, excessive or 
unexpected events immediately with the PI.  The PI and/or authorized study personnel 
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will review study charts and ongoing data collected on all participants on a weekly basis 
to ensure safety. In our small study, it is unlikely that use of statistics would be necessary 
to determine if excessive events were occurring; however, paired t-tests could be used if 
appropriate. We do not anticipate that there would be any specific events, other than the 
unexpected, that would trigger the suspension of our study. 
 
 
 
 
22.0 Compensation for Research Related Injury 
22.1 If the research involves more than minimal risk to subjects, describe the 
available compensation in the event of research-related injury, if any: 
 
Participants will not be provided any form of compensation for medical 
treatment or other damages (for example lost wages, time lost from work, 
etc.). If a participant becomes injured or sick from the research, they will 
be referred to a clinic or to their personal health care provider. Medical 
treatment may be provided at their expense or at the expense of their 
insurance company.  
 
22.2 Provide a copy of contract language, if any, relevant to compensation for 
research-related injury. At Virginia Tech, this is most common for 
sponsored research: 
 
N/A 
 
23.0 Economic Burden to Subjects 
23.1 Describe any costs that subjects might be responsible for because of 
participation in the research, including any uncompensated costs for items 
such as transportation, missed work, and childcare: 
 
The participant will be responsible for costs that may include purchase of 
athletic clothes or shoes to participate in the running and impact loading 
exercise sessions or the uncompensated cost that might include 
transportation, missed work, or childcare. 
 
24.0 Consent Process 
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24.1 Indicate the process by which you will obtain consent for study 
participation. Please upload all consent, parental permission, and assent 
forms, documents, and scripts referenced in this section to Protocol 
Management. 
Describe the following: 
● 
Where the consent process will take place (e.g., clinic waiting area, 
classroom, online) 
● 
The time interval between sharing the consent information with the 
prospective subject and obtaining consent. For lab, interview, and 
focus group studies, the Virginia Tech IRB prefers that subjects have 
at least 24 hours to review the consent form and study information 
before the appointment where consent will be obtained. For simple 
online survey studies, you can typically present the consent 
information immediately before subjects begin participation. 
● 
If applicable, processes to ensure ongoing consent or assent (e.g., 
for multiple sessions; for research in which a minor will turn 18 
during the study; for longitudinal research with minors who will 
later be asked to provide or affirm their assent). 
● 
Please review “SOP: Informed Consent Process for Research (HRP-
090)” for recommended procedure. Describe your process, being 
sure to include: 
o 
The name and role of all study personnel who will be trained 
and certified by the PI to conduct the consent process 
o 
The time that will be devoted to the consent discussion 
o 
Steps that you will take to minimize the possibility of coercion 
or undue influence 
o 
Steps that you will take to gauge or ensure the subjects’ 
understanding 
 
Participants will be first screened over the phone (phone script and 
screening form uploaded). The phone screening will include an overview 
of the study, the time commitment and the possible risks to participation. 
After explaining the study and before conducting the phone screen, a study 
team member will describe the purpose of the phone screen and what type 
of data will be collected, and then ask that the participant provide verbal 
permission to conduct the phone screening.  They will also be informed 
that they may refuse to answer any and all questions. An informed consent 
form will be emailed (or mailed) to the participant following the phone 
screening and at least 24 hours in advance of coming to the laboratory for 
the screening visit (email template uploaded).   
At the start of the screening visit, a team member will provide potential 
participants with a written copy of the study consent form and review the 
document with the participant. Participants will be encouraged to ask 
questions and seek clarification during the phone screening. The 
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participant will then be encouraged to ask questions before providing 
written consent. As much time as necessary will be devoted to address 
participant concerns.  Once the participant is ready to sign, she will be 
allowed to sign in a private room near the door where they may also exit 
the lab if they no longer wish to participate. Screening and informed 
consent will be performed by the doctoral student in charge of the study 
(T. Sterringer). Participants will be encouraged to ask questions and seek 
clarification during the phone screening and before signing the consent at 
the beginning of the laboratory screening visit. 
These steps including time to review the consent before the screening 
visit, time with study staff to review the protocol and address concerns, 
and time to sign the consent in a private setting and close to a laboratory 
exit will help minimize the possibility of coercion or undue influence.  
To help gauge the participant’s understanding, the team member will ask the 
participant to explain the study, when they need to notify the team about 
the start of menstruation, how often they will be asked to consume the 
meals and snacks provided by the research team, what foods and 
beverages they can consume outside of the ones provided by the research 
team, what physical activities they can do outside of the ones conducted in 
the NEM lab, and how often they would need to come to the lab to run.   
Non-English Speaking Subjects 
● 
Indicate what language(s) other than English are understood by 
prospective subjects or representatives. 
● 
If non-English speakers will be recruited, describe the process you 
will use to ensure that the oral and/or written consent information 
provided will be in a language that they understand.  
● 
If you translate consent forms and study materials, please provide a 
certified translation of the form as well as the certification 
document. 
● 
Indicate the spoken language that study personnel obtaining consent 
will use. Describe how you will assess fluency of personnel obtaining 
consent to ensure that the translation is accurate. 
 
 
Only English-speaking participants will be recruited for the study. 
 
 
Waiver or Alteration of Consent Process (consent will not be obtained, 
required information will not be disclosed, or the research involves 
deception) 
● 
Review the “CHECKLIST: Waiver or Alteration of Consent Process 
(HRP-410)” to ensure you have provided sufficient information for 
the IRB to make these determinations (i.e., that it meets the criteria 
for a waiver or alteration of the consent process). 
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N/A 
 
 
Subjects who are not yet adults (minors: infants, children, teenagers) 
● 
Describe the criteria that you will use to determine  legal age for 
consent to treatments or procedures involved in the research under 
the applicable law of the jurisdiction in which the research will be 
conducted (e.g., in Virginia, individuals under the age of 18 years). 
o 
For research conducted in Virginia, review “SOP: Legally 
Authorized Representatives, Minors, and Guardians (HRP-
013)” to determine which individuals in the state meet the 
definition of “minor.” 
o 
For research conducted outside of the state, please describe 
the legal requirements for the definition of “minor.”  
● 
Describe the process for obtaining parental permission.  
o 
Permission from one parent is acceptable for studies that 
involve no greater than minimal risk OR involve greater than 
minimal risk but present the prospect of direct benefit to the 
minor subject. 
o 
Permission from both parents is required in all other 
cases (unless one parent is deceased, unknown, 
incompetent, or not reasonably available, or when only 
one parent has legal responsibility for the care and 
custody of the minor). 
● 
Describe whether you will obtain permission from individuals other 
than parents or Legally Authorized Representatives, and if so, who 
will be allowed to provide permission. Describe the process you will 
use to determine these individuals’ authority to consent to the 
minor’s general medical care. 
● 
Indicate whether you will obtain assent from all, some, or none of 
the minors. If you will obtain assent from some minors, indicate 
which minors will be required to assent. Consider chronological age 
and intellectual capacity when determining who will be required to 
provide assent (e.g., infants are unable to assent. However, 
teenagers are likely able to read and sign an assent form). 
● 
When assent of minors is obtained, describe whether and how you 
will document it.  Will minors sign an assent form or give verbal 
assent? 
● 
Attach parental permission and minor assent forms or scripts in 
Protocol Management.  
 
 
N/A 
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Adults Unable to Consent  
● 
Describe the process you will use to determine whether an individual 
adult is capable of consent. 
● 
List the individuals from whom you will obtain permission in order 
of priority (e.g., durable power of attorney for health care, court 
appointed guardian for health care decisions, spouse, and non-
minor child). 
o 
For research conducted in the Virginia, review “SOP: Legally 
Authorized Representatives, Minors, and Guardians (HRP-
013)” to determine which individuals in the state meet the 
definition of “legally authorized representative.” 
o 
For research conducted outside of Virginia, please describe 
the legal requirements for obtaining permission from a legally 
authorized representative in the state where the research will 
occur. 
● 
Describe the process for assent of the subjects. 
o 
Indicate whether you will require assent from all, some, or 
none of the subjects. If some, indicate which subjects will be 
required to assent and which will not. 
o 
If you will not obtain assent from some or all subjects, please 
provide justification for not obtaining assent. 
o 
Describe whether and how you will document assent.  
N/A 
25.0 Process to Document Consent in Writing 
25.1 Consult “SOP: Written Documentation of Consent (HRP-091)” for 
recommended procedures, and describe whether and how consent of the 
subject will be documented in writing: 
 
Individuals who respond to the advertisements will be contacted by phone where they 
will be informed of the general plan of the study and all specific procedures included in 
the study (previously outlined in section 12). Participants will then be given a chance to 
ask questions regarding study procedures and risks.  Those still interested will be 
screened over the phone to determine eligibility based on current training status, running 
frequency and volume, dietary restrictions, medication use, injury history, and other 
criteria outlined in section 12.2 (see uploaded Screening form). Eligible individuals will 
be sent a copy of the consent form via email to review prior to coming to the lab.  They 
will then be given a chance to ask any questions either by email or during their scheduled 
screening/baseline visit. Those still interested will be asked to sign the consent during 
their first visit, before any data is collected. This information is detailed in section 24 
above. A copy of the informed consent will be sent to all participants.  
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25.2 If the research presents no more than minimal risk of harm to subjects and 
involves no procedures for which written documentation of consent is 
normally required outside of the research context, you can request that the 
IRB waive the requirement to obtain written documentation of consent 
(e.g., consent to participate is indicated by pressing a button for an online 
questionnaire – after the consent information is presented and before the 
questionnaire begins): 
 
Waiver of written consent to perform the phone screening is requested.  
The phone screening form has been uploaded.  
 
25.3 If you will document consent in writing, attach a consent document with 
places for signatures. If you will obtain consent, but not document consent 
in writing, please attach the consent script or text. Review “CHECKLIST: 
Waiver of Written Documentation of Consent (HRP-411)” to ensure that 
you have provided sufficient information. You should use “TEMPLATE 
CONSENT DOCUMENT (HRP-502)”to create the consent document or 
script: 
 
See the attached participant consent form 
 
26.0 Resources Available 
26.1 Describe the resources available to conduct the research. For example, as 
appropriate: 
● 
Describe the PI’s availability to supervise the research. 
● 
Justify the feasibility of recruiting the required number of suitable 
subjects within the agreed recruitment period. For example, how 
many potential subjects do you have access to? What percentage of 
those potential subjects do you need to recruit? 
● 
Describe the time that you will devote to conducting and completing 
the research. 
● 
Describe your facilities. 
● 
Describe the availability of medical or psychological resources that 
subjects might need as a result of an anticipated or unanticipated 
consequence of participation in the research. 
● 
Describe your process to ensure that all persons assisting with the 
research are adequately informed about the protocol, the research 
procedures, and their duties and functions (e.g., training plans, 
detailed study notebooks). 
 
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The PI is a Professor in the Department of Human Nutrition, Foods and Exercise at 
Virginia Tech. She currently has a 33% research appointment and oversees four doctoral 
students. She has previously served as a research dietitian and research exercise scientist 
at the National Institute of Diabetes & Digestive & Kidney Diseases in Phoenix and the 
Pennington Biomedical Research Center in Baton Rouge, LA, respectively, and has 
experience conducting exercise training studies and controlled feeding trials.  The PI will 
dedicate time to this study and ensure the doctoral student is adequately trained and 
performs all aspects of the study according to protocol and procedures. The doctoral 
student, Trisha Sterringer, will be in charge of participant recruitment and enrollment, 
protocol execution, data collection and the day-to-day aspects of the study. T. Sterringer 
is a registered dietitian (RD), NSCA certified strength and conditioning specialist, and 
ISCD Certified Bone Densitometry Technologist. She has previous experience 
conducting energy availability studies in competitive athletes at Case Western Reserve 
University involving indirect calorimetry, and assessments of dietary intake, exercise 
energy expenditure, and body composition. Trisha Sterringer will be the doctoral student 
in charge of the study as part of her PhD dissertation, however, additional undergraduate 
research assistants may be added to the study in the future. Dr. Elaina Marinik has 
extensive experience in study management and coordination at Virginia Tech and will be 
involved in the study to assist as needed. There are several key laboratories in the HNFE 
Department that will be utilized for this proposed study as described below. 
The Nutrition and Exercise Metabolism (NEM) Laboratory is directed by DE 
Larson-Meyer and located on the Virginia Tech Corporate Research Center campus. 
Major equipment items in the NEM Laboratory include Parvomedics TrueOne 2400 
metabolic cart, private room with a table for completing questionnaires, Woodway 
treadmill, and refrigerator and freezer storage. Free parking is available on-site and 
restrooms, showers, and changing facilities are also available.   
The Laboratory for Eating Behaviors and Weight Management (Director: Brenda 
Davy) is located in Wallace Hall and encompasses a ~600 sq ft Metabolic Kitchen with a 
~900 sq ft research Dining Laboratory area and a research dietitian computer workstation 
(for dietary analysis software), reach-in freezer, and refrigerator for storing meals to be 
consumed off-site, and an additional ~250 sq ft space housing stadiometers, scales, tables 
for completing questionnaires, a private room for measuring anthropometrics, and a file 
storage area. Also located in Wallace Hall 233 is a Lunar iDXA (GE Healthcare) and 
space for sample processing. 
 
The Human Integrative Physiology Laboratory is located on the Corporate 
Research Campus at the Garvin Innovation Center. The major equipment items in this 
laboratory include Lunar Prodigy DXA (GE Healthcare) and space for sample processing 
(i.e., wet lab areas). Additional research space is also available in Wallace Hall for 
sample processing (i.e., wet lab areas) and storage (-80 freezers).  
 
The Metabolic Core at Virginia Tech will perform the majority of the biochemical 
analyses. This core laboratory is housed in the Integrated Life Sciences building and 
includes a 140 sq ft. laboratory space dedicated to biochemical assays. This Core 
laboratory has a BioTek Synergy 2, a multi-mode microplate reader equipped with Gen5 
software capable of measurements of absorbance utilizing a monochrometer for 
wavelength selection from 200nm to 999nnm, and for fluorescence with excitation and 
emission filters for luminescence using a liquid-filled light guide. 
Virginia Tech Institutional Review Board Protocol No. 22-168 
Approved September 11, 2023
48 of 49

22-168 The Effect of Impact Loading on Bone Biomarkers in Energy-Restricted Female 
Runners 
VT Research Protocol version 1.0.1 
Page 49 of 49 
Revised: March 5, 2019 
 
27.0 Multi-Site Research 
Contact the HRPP for multi-site research (involving multiple institutions) and 
the details required for this section will be provided. Otherwise, indicate N/A. 
 
N/A 
Virginia Tech Institutional Review Board Protocol No. 22-168 
Approved September 11, 2023
49 of 49