Creatine Monohydrate Use and Muscle Stretching in Peripheral Artery Disease
Phase: PHASE2
Conditions: Peripheral Arterial Disease
Sponsor: Florida State University

 
 Cover Page  
Study Title: Creatine Use and Muscle Stretching in Peripheral Artery Disease  
NCT Number: NCT04471792  
Date of Study Protocol: 03-09-2022  
Study Protocol to Follow 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
Page 1 of 19 
Revised: December 1, 2018 
PROTOCOL TITLE: 
Creatine supplementation and muscle stretching in peripheral artery disease patients  
PRINCIPAL INVESTIGATOR: 
Judy Muller-Delp, PhD 
 
Biomedical Science  
850-567-8056 
Judy.Delp@med.fsu.edu 
VERSION NUMBER/DATE: 
Version 6 (3/9/2022) 
 
REVISION HISTORY 
 
Revision 
# 
Version Date 
Summary of Changes 
Consent 
Change? 
1 
11/7/19 
Added use of Magnetic Resonance Imaging 
Yes 
2 
3/16/20 
Research device (section 2.1), renal disease 
(section 10.2 (exclusion); page 2&4 of 
consent), conflict of interest (consent form) and 
recruitment language (section 13.0). 
Yes 
3 
5/01/20 
Ability to consent patients at VSA 
No 
4 
7/1/20 
Changed Dr. Delp to PI; Increased subjects to 
50; clarified inclusion criteria for healthy 
volunteers; changed age range 40-95; Added 
demographics, vitals, SF36, WIQ, and iPAQ to 
visit 2; added 5 day study visit window; limited 
MRIs to 4-6 healthy and 6-8 PAD in each 
supplement group; added diary for splint 
stretching and supplement use tracking; added 
REDCap for data management. 
Yes 
5 
8/25/21 
Added SF-36, WIQ, and IPAQ to visit 3. 
No 
6 
3/9/21 
Added a Fitbit to visit 1 & 3 to monitor step 
count (section 6.2).  Added 8 foot up and go 
test, 30 second chair rise test, and chair sit and 
reach to visit 2 & 3 (section 6.2).  Added heel 
raises for 1 minute with NIRS to visit 2 & 3 
(section 6.2).  These changes are also reflected 
in the informed consent document (pages 5-7).  
Changed estimated visit length in the consent 
form from 2-3 hours to 2-3.5 hours to reflect 
addition of new tests. 
Yes 
 
 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
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Revised: December 1, 2018 
Table of Contents 
1.0 
Study Summary ........................................................................................................ 3 
2.0 
Objectives* .............................................................................................................. 4 
3.0 
Background* ............................................................................................................ 4 
4.0 
Study Endpoints* ..................................................................................................... 5 
5.0 
Study Intervention/Investigational Agent ................................................................ 5 
6.0 
Procedures Involved* ............................................................................................... 6 
7.0 
Data and Specimen Banking* ................................................................................ 10 
8.0 
Sharing of Results with Subjects* ......................................................................... 10 
9.0 
Study Timelines* ................................................................................................... 10 
10.0 Inclusion and Exclusion Criteria* .......................................................................... 10 
11.0 Vulnerable Populations* ........................................................................................ 12 
12.0 Local Number of Subjects ..................................................................................... 12 
13.0 Recruitment Methods ............................................................................................. 12 
14.0 Withdrawal of Subjects* ........................................................................................ 12 
15.0 Risks to Subjects* .................................................................................................. 13 
16.0 Potential Benefits to Subjects* .............................................................................. 15 
17.0 Data Management* and Confidentiality ................................................................ 16 
18.0 Provisions to Monitor the Data to Ensure the Safety of Subjects* ........................ 17 
19.0 Provisions to Protect the Privacy Interests of Subjects .......................................... 17 
20.0 Compensation for Research-Related Injury ........................................................... 18 
21.0 Economic Burden to Subjects ................................................................................ 18 
22.0 Consent Process ..................................................................................................... 18 
23.0 Process to Document Consent in Writing .............................................................. 19 
24.0 Setting .................................................................................................................... 19 
25.0 Resources Available ............................................................................................... 19 
26.0 Multi-Site Research* ............................................................................................. 19 
 
 
 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
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Revised: December 1, 2018 
1.0 
Study Summary 
 
Study Title 
Creatine supplementation and muscle stretching in peripheral 
artery disease patients 
Study Design 
Randomized, single blind, cross sectional 
Primary Objective 
To utilize near-infrared spectroscopy and magnetic-
resonance imaging to investigate if stretch training, when 
combined with creatine loading improves the 
cardiovascular performance in PAD patients 
Secondary 
Objective(s) 
To characterize near-infrared spectroscopy derived tissue 
oxygenation responses at rest and during a walking test 
(i.e., six-minute walk test).  
Research 
Intervention(s)/ 
Investigational 
Agent(s)  
Creatine Monohydrate 
IND/IDE #  
IND 145110 
Study Population 
Individuals aged 40 - 95 
Sample Size 
Up to 50 
Study Duration for 
individual 
participants 
About 5-6 weeks 
Study Specific 
Abbreviations/ 
Definitions  
6MWT: six-minute walk test 
NIRS: Near-infrared spectroscopy  
PAD:  Peripheral Artery Disease 
 
 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
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Revised: December 1, 2018 
2.0 
Objectives* 
2.1 
1. To utilize near-infrared spectroscopy to investigate if our 
research device for stretch training, and creatine loading impact 
cardiovascular performance in aged and PAD patients.  
2. To characterize near-infrared spectroscopy derived tissue 
oxygenation responses at rest and during a walking test (i.e., six-
minute walk test). We will also assess parameters with Magnetic 
Resonance Imaging.  
2.2 
It is hypothesized that the stretching protocol will improve both 
NIRS derived tissue oxygenation and magnetic resonance derived 
leg blood flow measures and that creatine supplementation will 
further improve phosphorus metabolite muscle performance. All 
patients will undergo either 4 weeks of stretch training with- or- 
without creatine supplementation according to previously defined 
creatine guidelines (Sanchez-Gonzalez et al 2011). 
3.0 
Background* 
3.1 
Lower extremity peripheral artery disease (PAD) has been 
estimated to impact nearly 8.5 million U.S. adults above the age 
of 40, significantly increasing the rate of morbidity and mortality 
with concomitant decreases in quality of life. These patients are 
often given medical therapy (e.g., statins, antiplatelet, 
anticoagulants) and are also recommended to begin structured 
exercise programs. However, limb ischemia is common in these 
patients during physical activity which may limit exercise 
tolerance. Specifically, (Bauer, Brass, & Hiatt, 2004; 2011) have 
shown that a major limitation in PAD patients is an impaired 
muscle metabolism. These data are important in that they show 
alterations in blood flow and metabolic machinery likely impact 
exercise tolerance. As such, development of tolerable 
countermeasures to improve limb blood flow and muscle 
energetics may increase adherence to exercise therapy and 
improve health outcomes in PAD patients. Our lab has previously 
shown that daily stretching, 30-minutes of dorsiflexion, 
significantly improved soleus muscle function and muscle blood 
flow during exercise in a rat model of aging (Hotta et al., 2018). 
In a follow up study, we have also shown that this model 
improves vascular function in human PAD patients (Hotta et al; in 
review). As noted above, muscle energetics are delayed in PAD 
patients, so improving the rest-to-exercise transition, with 
creatine supplementation, and muscle blood flow may help PAD 
patients sustain exercise longer. 
3.2 
Our lab has previously shown in animal models and human that 
muscle stretching improves leg blood flow Hotta et al. (2018, 
2019). We are now testing to see if an added supplement can 
further improve these findings. 
 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
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Revised: December 1, 2018 
4.0 
Study Endpoints* 
4.1 Improvement in muscle oxygenation, shown via near-infrared 
spectroscopy, and blood flow/phosphorus metabolites, via magnetic 
resonance imaging. 
4.2 Improvement in walking distance, shown via 6MWT. 
5.0 
Study Intervention/Investigational Agent 
5.1 Creatine Monohydrate (Cr) (C4H11N3O3) is a dietary supplement 
commonly found in red meat and seafood (3). Cr is given in doses 
ranging from 2grams/day up to 30 grams/day for up to 5 years safely 
(1). Previous work suggests that an initial 5 day loading period 
(10g/day) followed by a maintenance phase (5 g/day) be followed to 
obtain maximal response (3). The objectives of the current pilot 
study are to investigate if Cr supplementation plus muscle stretching 
improves 6MWT distances in patients with peripheral artery disease 
(see (1) for review). The route of administration for the Cr is by 
mouth. With over 1,000 studies using Cr, the safety and efficacy of 
Cr are very consistent with the main side effect being weight gain (2, 
3). The use of Cr even extends to ischemic heart disease, aging, and 
neurodegenerative disease. Currently, there are no studies suggesting 
adverse effects that would contraindicate use in patients with 
peripheral artery disease 
5.2 Drug/Device Handling: The researchers will purchase the creatine 
monohydrate from GNC supplements. The creatine containers will 
be stored in the laboratory and remain unopened until the patient 
takes them home. This supplement has been given full approval by 
the FDA (See IND above). 
1. Hotta K, Behnke BJ, Arjmandi B, Ghosh P, Chen B, Brooks R, Maraj JJ, Elam ML, Maher P, Kurien 
D, Churchill A, Sepulveda JL, Kabolowsky MB, Christou DD, and Muller-Delp JM. Daily muscle 
stretching enhances blood flow, endothelial function, capillarity, vascular volume and connectivity 
in aged skeletal muscle. J Physiol 596: 1903-1917, 2018. 
2.  Hotta, K., Batchelor, W. B., Graven, J., Dahya, V., Noel, T. E., Ghai, A., . . . Muller-Delp, J. (2019). 
Daily Passive Muscle Stretching Improves Flow-Mediated Dilation of Popliteal Artery and 6-minute 
Walk Test in Elderly Patients with Stable Symptomatic Peripheral Artery Disease. Cardiovasc 
Revasc Med, 20(8), 642-648. doi:10.1016/j.carrev.2019.05.003 
3. Hultman E, Soderlund K, Timmons JA, Cederblad G, and Greenhaff PL. Muscle creatine loading in 
men. J Appl Physiol (1985) 81: 232-237, 1996. 
4. Kreider RB, Kalman DS, Antonio J, Ziegenfuss TN, Wildman R, Collins R, Candow DG, Kleiner SM, 
Almada AL, and Lopez HL. International Society of Sports Nutrition position stand: safety and 
efficacy of creatine supplementation in exercise, sport, and medicine. J Int Soc Sports Nutr 14: 18, 
2017. 
5. Shao A, and Hathcock JN. Risk assessment for creatine monohydrate. Regul Toxicol Pharmacol 45: 
242-251, 2006. 
 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
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Revised: December 1, 2018 
5.3 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: 
• 
Judy Muller-Delp. 
• 
21 CFR 11: Data collection/security parameters are outlined in 
the data management section of this protocol.    
• 
21 CFR 54: FDA COI forms will be completed as required. 
• 
21 CFR 210: All creatine monohydrate and placebo 
supplementation will comply with good manufacturing practice 
regulations. 
• 
21 CFR 211: GNC and Allergy Research Group both comply 
with the good manufacturing regulations. 
• 
21 CFR 312: We have submitted an IND and have approval for 
the use of creatine monohydrate in this investigation.  
 
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 
 
 
6.0 
Procedures Involved* 
6.1 
All patients (PAD or healthy volunteers) will undergo 4 weeks of splint 
stretching (30min/day; 5days/week – procedures attached with this 
submission) and be randomized into groups with or without creatine 
supplementation (10g/day for the first 5 days and 5g/day for the 
remainder). Placebo supplementation (Fiber powder; cellulose) will be 
given during the opposite arm of the study and according to section 6.2 
(below).  
6.2 
Each potential patient/participant will be screened to determine potential 
eligibility by the research team. If the individual is eligible, see 
inclusion/exclusion criteria, they will be asked to consider joining the 
study. Individuals meeting the initial criteria and who agree to participate 
in the study will sign informed consent and complete any medical history 
forms. All participants must abstain from alcohol for 24 hours and 
caffeine/food for ~ 4 h prior to testing sessions. The current investigation is 
a randomized single-blind study design. 
• C1. Health and exercise questionnaires: Subjects’ medical 
history and exercise habits will be  

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
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assessed by self-reported health and exercise questionnaires: 
demographics and health history (Visit 1), physical activity 
readiness (Visit 1), health survey-SF36 (Visit 2 and 3), 
international physical activity questionnaire (I-PAQ, Visit 2 and 
3), walking impairment questionnaire (WIQ, Visit 2 and 3).    
• C2. Fitbit: Subjects will be given a Fitbit Charge 2 (Fitbit, San 
Francisco, California) to wear for a 1 week period starting at 
Visit 1 and Visit 3 to measure daily step count before and after 
the intervention.  At this time, we anticipate 4-8 subjects with 
PAD using a Fitbit during the course of the study. We will 
recruit 2-4 PAD  patients to serve as time controls and not 
partake in the intervention. 
• C3. 'Anthropometric measurements and vitals will consist of 
subject height measured on a stadiometer and weight measured 
on a calibrated scale, heart rate (resting) and baseline blood 
pressure after 10-min of quite supine rest. Next, we will measure 
circumference of the waist and lower leg and range of motion of 
the knee and ankle. All of these measurements will be done at 
Visit 2.  Resting heart rate and blood pressure will be repeated 
at Visit 3.    
• C4. `Muscle oxygenation characteristics' of the lower leg (i.e., 
gastrocnemius and soleus) will be measured via near-infrared 
spectroscopy (NIRS) (Moxy monitoring systems, Hutchinson, 
Minnesota) at Visit 2 and 3. NIRS is a completely noninvasive 
measure that uses infrared light waves emitted from a skin 
surface probe. NIRS measurements will be made continuously 
during the entirety of the experimental protocol. Protocol one 
will utilize a blood pressure cuff around the knee joint and 
inflated to ~250mmHg to occlude flow for 5-minutes. At minute 
5 the cuff is instantaneously released and rest will continue for 
~5-minutes. The NIRS will be take off for C3.A and then put 
back on in place to complete C4 (see below). 
• C5.A ‘Muscle blood flow and metabolic kinetics” (i.e., 
gastrocnemius and soleus) will be measured in the 3T MRI 
system (Visit 2 and Visit 3; limited subjects). All patients and 
controls will be placed in the MRI feet first and undergo each 
trial by placing a blood pressure cuff just proximal to the knee 
joint and inflating it to ~250mmHg to occlude flow for 5-
minutes. At minute 5 the cuff is instantaneously released and rest 
will continue for ~5-minutes. After a brief rest period, the 
patient’s foot and ankle will be positioned in the splint device to 
produce dorsiflexion and stretch of the calf muscles and 
scanning will resume. Due to associated MRI costs, only a subset 
of patients and controls will be using the MRI. At this time, we 
anticipate ~25 total subjects will undergo pre/post MRI: 4-6 
healthy/placebo; 4-6 healthy/creatine; 6-8 PAD/placebo; 6-8 
PAD/creatine.  Subjects who undergo MRI will be selected in 

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disease patients 
 
 
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the order they present and are eligible to receive an MRI (based 
on MRI exclusion criteria)  
 
C6. ‘Cardiovascular assessment’ will be measured via the “6-
minute walk test (6MWT)” (Visit 2 and Visit 3) a sub-maximal 
clinically relevant test to assess efficacy of the 4-week 
intervention. All patients/participants will undergo a 6MWT 
pre/post intervention. Briefly, a standardized model will be 
followed according to American Heart Association guidelines. 
The 6MWT will be performed on a level surface by a research 
team member. Patients will be instructed to cover the greatest 
distance they can in 6-minutes at a pace of their choosing. Chairs 
will be placed periodically along the hallway in case a patient, or 
participant, needs to rest until they can begin again. The 6MWT 
data collection form and corresponding rating scales are 
provided with this submission. 
• C7. “Physical function” will be measured via the 8 foot up and 
go, 30 second chair rise, and chair sit and reach tests.  The 8 foot 
up and go requires the subject to start in a seated position, stand 
up, walk 8 feet, then return to the seated position.  The score for 
this assessment will be the number of seconds needed to 
complete this task.  The subject will perform this assessment 
twice, and the best score of the two trials will be used.  Next, the 
subject will perform the 30-second chair stand test.  In this 
assessment, the number of full stands from a seated position will 
be counted in the 30-second time period.  The chair sit and reach 
will require the subject to reach his/her fingertips towards a 
straightened leg from a seated position.  This assessment will be 
performed three times on each leg.  The score will be the number 
of centimeters between the fingertips and toes for each leg. 
• C8. “Muscle oxygenation characteristics” of the lower leg (i.e., 
gastrocnemius and soleus) will also be evaluated via NIRS with 
a heel raise test.  The subject will complete heel raises for a 1 
minute period while we continuously monitor muscle 
oxygenation during exercise and recovery. 
• C9. ‘Creatine supplementation and placebo control’ will consist 
of an initial loading dose (see consent) of 10g/day for 5 days and 
then a daily 5g/day for 4 weeks. All creatine and placebo 
powders will be given in containers marked with a date for 
consumption and serving spoon and instruction guide. All 
creatine will be purchased from a local nutrition store (General 
Nutrition Center, GNC). All cellulose will be purchased from an 
online supplement store (Allergy Research Group, ARG). 
Subject instructions for taking the supplement will be provided 
to them and are attached to this submission.  
• Subjects’ visits (after screening visit) are planned for baseline 
(Visit 2) and Day 28 (Visit 3).   However, due to scheduling 

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conflicts that are likely to arise, if a subject cannot come back on 
Day 28, there will be a 5-day study visit window (+/-) to allow 
for scheduling but not acquire protocol deviations. Stretching 
and supplementation will continue to occur during that time. Any 
study visit outside of this extended visit window will be 
documented as a deviation. Finally, any subject/patient that 
doesn’t adhere to a “no alcohol for 24h or food/caffeine 4h” 
prior to testing will be rescheduled for another later date.  
6.3 Describe: 
Specific risks:  
Healthy control subjects and peripheral artery disease patients: 
uncomfortable muscle stretch and blood pressure cuff. Fitbit may 
cause skin irritation. 
Placebo: uncomfortable muscle stretch, blood pressure response to 
walking. Bloating, gas, diarrhea from fiber pill.  
Creatine: uncomfortable muscle stretch, blood pressure response to 
walking. Bloating, gas, diarrhea, muscle cramping during creatine 
supplementation. We are unaware of any further risks with creatine 
supplementation. 
We will reduce risk of patients with a standardized protocol across all 
subjects. For example, we will maintain weekly contact with the 
patients both inside the laboratory and while at home with phone calls 
and messages. In addition, the cardiologist's knowledge of the patient 
will provide us with the information to include or exclude the patient. 
During the most vigorous task (six-minute walk test) we will place 
chairs along the hallway in case the patient gets too tired to continue 
and can rest until they are recovered. 
To reduce the risk of skin irritation associated with the Fitbit, all subjects will 
be instructed to keep the device clean, dry, and to not wear the device too 
tightly.  If skin irritation occurs and does not resolve within 3 days, subjects 
will be instructed to discontinue use of the device.  If any soreness, tingling, 
numbness, burning, or stiffness in the hands occurs in the hands or wrists, 
subjects will be instructed to discontinue use of the device immediately.  To 
reduce risk of the above, subjects will be given the following instructions:  
 
1. Do not attempt to replace the battery, open the enclosure or 
disassemble your Fitbit product.  
2. Do not use your Fitbit product if the display is cracked. 
3. Do not expose your Fitbit product to extremely high or low 
temperatures. 
4. Do not use your Fitbit product in a sauna or steam room. 

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disease patients 
 
 
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5. Do not use abrasive cleaners to clean your Fitbit product. 
6. Remove your Fitbit product immediately if it feels warm or hot. 
7. Charge the battery with the charging cable provided.  Do not use other 
cables. 
8. Do not wear your Fitbit product while charging it. 
9. Do not charge your Fitbit product while it is wet. 
 
Creatine monohydrate will be used in the current investigation. The 
purpose of using creatine is to create a favorable metabolic 
environment to improve patients’ ability to walk. 
• The use of creatine monohydrate in this investigation has been 
approved by the FDA. 
The source records that will be used to collect data about subjects are 
attached to the IRB submission and will be kept in a locked cabinet 
in a locked office. 
6.4 All data will be collected by trained members of the study team 
(NIRS, MRI, blood pressure, heart rate, anthropometry, Fitbit, and 
cardiovascular (i.e., six-minute walk test) variables.  
6.5 N/A 
6.6 N/A 
7.0 
Data and Specimen Banking* 
7.1 N/A 
8.0 
Sharing of Results with Subjects* 
8.1 Deidentified data will be provided for the 6MWT and the tissue 
oxygenation if the patient asks. These results will not include their 
name or any way to identify the patient.  
9.0 
Study Timelines* 
9.1 
All subjects will be asked to come to 3 visits (Visit 1: initial 
screen, paperwork and surveys; Visit 2: pre-testing and surveys; Visit 3: 
post-testing). Day one is approximated to take 30-45 minutes. Testing 
days two and three are expected to take up to ~2.5 hours. The whole study 
will take 5-6 weeks to complete.  
9.2 
We estimate 6 – 12 months for subject enrollment.  
9.3 
We anticipate complete primary analysis by summer 2021 
10.0 Inclusion and Exclusion Criteria* 

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10.1 The inclusion criterion for the PAD study group is a resting ankle-
brachial index (ABI) of 0.90 or less in either leg or clinical diagnosis 
by a medical doctor. All patients must have a stable condition for at 
least 3 months. Subjects in the healthy volunteer group do not need 
to meet the PAD inclusion criteria. 
• Subjects must be ages 40 - 95 
• PAD patients will be screened/cleared to participate by 
their primary care physician/cardiologist and then by our 
research team. Healthy volunteers do not require physician 
clearance to participate. 
• All subjects must have the ability to speak and read English 
to be included in this study.   
10.2 Subjects (PAD and healthy controls) are excluded according to 
whether they meet one or more of the following criteria: • habitual 
exercise or cardiovascular rehabilitation program during the past 3 
months • below or above-knee amputation, critical limb ischemia 
(ulceration or gangrene) • leg pain at rest • major surgery or lower 
extremity revascularization during the previous 3 months • major 
medical illness treatment during the prior 12 months • central 
neurological disease • limited ankle or knee joint range of motion • 
requirement of oxygen with activity or exercise • Any New York 
Heart Association level of heart failure • atrial fibrillation • 
wheelchair confinement, or inability to walk • cognitive disorder • 
vasculitis problem including Takayasu’s arteritis, Berger’s disease, 
collagen disease or Raynaud’s disease • Subjects whose physician 
feels that participation in this research project would not be 
appropriate for him/her will be excluded. • overt cardiovascular 
disease • metabolic disease • renal disease 
Exclusion specific to the MRI: If patients have any history of 
pacemakers or pacer wires, open heart surgery, artificial heart 
valves, aneurysm clips, cochlear implants, braces or extensive dental 
work, implanted electrical or mechanical devices, tissue expanders, 
foreign metallic objects from explosives, shrapnel or metalwork 
fragments, or artificial limbs. Subjects will also be excluded if they 
are pregnant, claustrophobic, have tremors or cannot lie still for 1-2 
hours. 
If subjects only have exclusion criteria listed in the MRI section, they can 
be included in the study, but will not participate in the MRI portion at 
Visit 2 and Visit 3. 
• 
This investigation will not include:  
• 
Adults unable to consent 

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• 
Individuals who are not yet adults (infants, children, teenagers) 
• 
Pregnant women 
• 
Prisoners 
11.0 Vulnerable Populations* 
11.1 N/A 
12.0 Local Number of Subjects 
12.1 The goal of this study is to enroll 20 individuals with PAD and 20 
healthy volunteers. To account for screenfails and withdrawals, we 
will enroll up to 50 subjects in this study. 
12.2 N/A 
13.0 Recruitment Methods 
13.1 Subjects/patients will be identified based on required demographics 
(i.e. age) via word of mouth, email, flyers and visits to local clinics 
(e.g., cardiologists). We will also recruit by going to support groups, 
senior centers, or retirement homes in the area. These are not specific 
and we always contact the property to set up a meeting and get 
approval to be on their grounds to talk with residents. 
We have also partnered with Vascular Surgery Associates in 
Tallahassee, FL (VSA) to recruit PAD patients. Prior to any contact 
with VSA patients, applicable study team members will also sign 
related documents that are provided by the VSA legal department. 
VSA employees will assess patient records based on our set 
inclusion/exclusion criteria. The VSA will then contact potential 
subjects that match our inclusion/exclusion criteria. If they are 
interested in being in the study, we will speak to these individuals to 
explain the study and enroll them if possible. 
 
13.2 Source of the subjects, Tallahassee, FL and surrounding areas. 
13.3 All PAD patients will be identified by their primary care 
physician/cardiologist and our health history questionnaires. Each 
PAD patient must complete the consent form (with exclusion 
criteria) and obtain their physician approval or will not be included 
in the current investigation. Healthy controls will be identified by 
recruitment shown in 13.1 and consented to participate. 
13.4 Materials to recruit patients and controls have been uploaded. 
13.5 Each patient and control that completes all the testing will get 
$100.00 via cash or gift card. 
14.0 Withdrawal of Subjects* 

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14.1 Failure to comply with creatine/placebo use or stretching protocol or 
inability to attend study visits. 
• Meets any of the exclusion criteria 
14.2 The subject will be informed via telephone, email or in person that 
they have been removed from the study. We will also remind them at 
multiple time points that this study is completely voluntary and they 
may stop at any time.  
14.3 If the patient removes themselves from the investigation all data 
collected up to the point of withdrawal will be kept.  The reason for 
subject withdrawal will be recorded.    
15.0 Risks to Subjects* 
15.1 Specific risks:  
Healthy control and peripheral artery disease patients: uncomfortable 
muscle stretch and blood pressure cuff.  
Placebo: uncomfortable muscle stretch, blood pressure response to 
walking. Bloating, gas, diarrhea from fiber pill.  
Creatine: uncomfortable muscle stretch, blood pressure response to 
walking. Bloating, gas, diarrhea, muscle cramping during creatine 
supplementation. We are unaware of any further risks with creatine 
supplementation. 
• MRI: According to the FDA, there is currently no evidence that MRI 
with approved scanners of up to 4 Tesla signal strength are associated 
with adverse effects. Notwithstanding, there are several major 
potential risks. The first of these is discomfort that subjects may 
experience from being in a confined and sometimes noisy 
environment. Second, strong magnetic fields can have a deleterious 
effect on electronic, magnetic and metal devices that subjects may 
carry on them or have been implanted in their bodies. Third, if 
unprotected, hearing loss can occur with protracted exposure to the 
noise generated during MRI imaging. In addition, subjects may 
temporarily experience dizziness and/or nausea if they move rapidly 
through the magnetic field. There is also the potential for radio 
frequency (RF)-induced local tissue heating and for mild electrical 
stimulation due to induced currents as magnetic gradients switch 
rapidly. Known as peripheral nerve stimulation (PNS), this is typically 
not harmful but may cause discomfort. Subjects may also experience 
physical discomfort from lying on their backs for 1 – 1.5 hours in the 
scanner. 
• To mitigate these risks the following steps will be taken: 
• When running certain scan types 3T MRI scanners can generate sound 
on the order of 126 – 131 dB (Foster et al., 2000; Hattori et al., 2007). 

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The FDA has recommended that safe audible levels are no more than 
99 dB(A) (US Food and Drug Administration, 2014). Therefore, 
anyone in the magnet room while it is in operation is required to 
correctly wear earplugs. Subjects will be encouraged to move slowly 
when entering and exiting the magnetic field. The scanner bed is 
designed to move slowly in order to minimize the possibility of 
dizziness. RF-induced local heating is unlikely due to software 
requirements to enter the subject’s height and weight accurately to 
determine specific absorption rates of RF energy. Moreover, padding 
will be extensively used to ensure that the subject is not in contact with 
parts of the scanner where RF energy emanates.  Subjects are warned 
of the possibility of mild electrical stimulation and are monitored 
throughout the session by means of audio intercom. Exclusions will be 
made for the following: cardiac pacemaker, metal fragments in 
eyes/skin/body (shrapnel), history of being a metal worker/welder, 
history of eye surgery/eyes washed out because of metal, aortic 
aneurysm clips, prosthesis, by-pass surgery/coronary artery clips, 
hearing aid, heart valve replacement, subjects with an I.U.D. (birth 
control device), a shunt (ventricular or spinal), electrodes, metal 
plates/pins/screws/wires, or neuro/bio-stimulators (TENS unit), vision 
problems uncorrectable with lenses, claustrophobia, inability to lie 
still on one’s back for approximately 90 minutes, prior neurosurgery, 
older tattoos with metal dyes, unwillingness to remove nose, ear, 
tongue or face jewelry, and women who are pregnant or currently 
lactating. 
While in the scanner voice contact will be maintained at all times and 
subjects will be encouraged to report discomfort. Furthermore, 
well trained staff are on hand to watch for signs of distress, anxiety 
or fatigue and terminate the study should the subject exhibit signs 
of distress.  
Subjects will be provided with an emergency squeeze ball to use if 
they would like to terminate the scan and talk to subject personnel. 
Subjects can terminate their scanning session at any time if they 
wish. 
We will make every effort to train subjects properly prior to the MRI 
scan. For example, a mock scanner can be used to assess subject’s 
ability to tolerate confined spaces and lie on their backs. They can 
also be played noises typical of the scanner environment. Subjects 
will be asked questions about their fear of confined spaces during 
the screening stage to reduce the risk of recruiting claustrophobic 
subjects. However, should subjects become claustrophobic during 
study or for any reason be unable to remain in the scanner, the scan 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
Page 15 of 19 
Revised: December 1, 2018 
will be terminated and the participant expeditiously removed from 
the scanner.     
Risks of assessments: The main risks are that the interviews and 
questionnaires are time consuming. These assessments, however, 
rarely pose psychological risks for the study participants. 
Mitigation of risk of assessment: While most subjects will find the 
interviewing to be a positive experience, some may become anxious 
or tearful because of the personal nature of the questions. 
Interviewers will exercise caution and sensitivity and terminate the 
interview as necessary in order to protect the emotional well-being 
of the subject.   
 
Risk of breach of confidentiality: Breaches in confidentiality could 
have an negative impact, but protections are in place to minimize the 
risk of this happening.  
 
Mitigating the risks of breach of confidentiality: To minimize the 
possible risk of breach of confidentiality, each study participant will 
be assigned, upon enrollment in the study, a study ID code that will 
be used as an identifier for the subsequent research-related activity. 
All information collected, including self-reported questionnaires and 
imaging data are identified solely with the study ID without any 
personal identifiers. Only strictly anonymized data will be used for 
statistical analysis. Contact information for the subjects will be 
collected in order to reach them for appointment reminders and to be 
able to communicate with them weekly.  This information will be 
kept on a separate form accessible only to the research team 
members who will be contacting the subjects.  Access to the subject 
ID list will be limited to specific members of the research team.   
Research paper files will be kept in a locked office and only made 
available to qualified personnel for research purposes. All data will 
be entered into REDCap database and stored in a HIPAA compliant 
environment. No verbal or written information concerning a subject 
will be released to anyone without the express written authorization 
of the subject.  
15.2 N/A 
15.3 N/A 
15.4 N/A 
16.0 Potential Benefits to Subjects* 

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disease patients 
 
 
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Revised: December 1, 2018 
16.1 Benefits 
Healthy control: None.  
 
Placebo: increasing functional capacity and cardiovascular health 
outcomes.  
Creatine: increasing muscle blood flow, increasing functional 
physical capacity and cardiovascular health outcomes.  
Static muscle stretching has been shown to improve muscle blood 
flow. We believe that combining creatine supplementation with 
muscle stretching will enhance the efficacy of the treatment protocol, 
further enhancing cardiovascular function. 
17.0 Data Management* and Confidentiality 
17.1 Statistical analysis will include repeated measures ANOVA with 
Bonferroni post hoc analysis and paired samples t-test when 
applicable. Due to the nature of the current investigation (pilot 
study) we will be running a power analysis once the data set is 
collected. 
17.2 During this study, and after consent is obtained, medical history 
questionnaires will be filled out prior to the start of the study. 
Experimental measurements will be obtained for statistical analyses 
and for research purposes only, and data will be kept in strict 
confidence. No information will be given to anyone without written 
permission from the subject. Data will be entered into REDCap and 
stored in a HIPAA compliant environment. Confidentiality will be 
ensured by use of identification codes. All data, whether generated in 
the laboratory or in post-processing will be identified with an 
identification code unique to the subject. The master identification 
code list will be kept under lock and key access limited to the P.I. 
and necessary members of the study team. Any presentation or 
publication of the data will be done in cumulative fashion without 
identifiers.   
17.3 Our protocols include standard operating procedures for calibration 
of instrumentation and all data collection. 
17.4 Describe how data or specimens will be handled study-wide: 
• 
All data collected on paper will be identified only by subject 
ID.  Paper forms will be entered and/or uploaded into 
REDCap database which only the research team will have 
access to.  Subject contact information will be necessary, but 
this information will be stored separately with limited access to 
the study team members who must contact the subjects for AE 
tracking and appointment reminders. 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
Page 17 of 19 
Revised: December 1, 2018 
• 
Individual participant paper files will be stored in a locked 
cabinet in a locked office only accessible to the research team. 
Any data extracted from the REDCap database will be secured 
with password protection. Electronic communication between 
investigators will only involve anonymous information.   
 
All data forms and study specific information will be kept in a 
locked file cabinet and in a password protected capable 
computer database with access limited to only the P.I. and key 
personal of the research team. Confidentiality will be protected 
by utilizing a code number as the only identifier for each 
subject.  
 
The PI or study staff will review all data collection forms on an 
ongoing basis for data completeness and accuracy as well as 
protocol compliance. A check list will be used to verify 
compliance. The frequency of data review for this study differs 
according to the type of data and can be summarized as 
follows:  
Data Type: Subject accrual (including compliance with protocol 
enrollment criteria); Frequency: Weekly; Reviewer: PI  
Data Type: Status of all enrolled subjects, as of date of reporting; 
Frequency: Weekly; Reviewer: PI  
Data Type: Adverse Events* and rates (including out-of-range lab 
values); Frequency: Weekly; Reviewer: PI   
Data Type: Serious adverse events**; Frequency: Per occurrence; 
Reviewer: PI *An adverse event (AE) is any untoward medical 
occurrence in a subject during participation in the study or with use 
of the experimental agent being studied. 
Upon completion of the study, the data will completely de-identified 
so that it may be kept indefinitely.  Once publications have been 
approved we will transfer all paper data into the FSU online system 
that is password protected. 
All study records must be kept in compliance with FDA regulations. 
18.0 Provisions to Monitor the Data to Ensure the Safety of Subjects* 
• 
We will not use data to monitor safety of subjects/patients. We 
are doing this by talking with the patients and subjects weekly.  
19.0 Provisions to Protect the Privacy Interests of Subjects 
19.1 Only the PI and the research team will have full access to patient 
records. All data will be deidentified when post processing. This 

PROTOCOL TITLE: Creatine supplementation and muscle stretching in peripheral artery 
disease patients 
 
 
Page 18 of 19 
Revised: December 1, 2018 
investigation is not to be kept in secret. All patients may talk about 
the study and study components to whomever they choose.  
19.2 We will be sure to inform the patients that this study is totally 
voluntary, and they are not required to answer anything they feel is 
uncomfortable or they do not want to answer. 
19.3 We will let the patients know their data will be coded and locked 
away with a similar level of security as a doctor’s office. Besides the 
PI and research team, no other lab help will  have sensitive 
information (i.e., health history) or any information to link the data 
back to the subject. 
20.0 Compensation for Research-Related Injury 
20.1 N/A 
20.2 N/A 
21.0 Economic Burden to Subjects 
21.1 N/A 
22.0 Consent Process 
22.1 Indicate whether you will you be obtaining consent, and if so 
describe: 
• 
Consent process will take place in the College of Medicine 
and/or at Vascular Surgery Associates.  
• 
Patients must first have doctor’s approval prior to second visit 
and consent. 
• 
Every week we will speak with patients and make sure they are 
comfortable and without any distress. 
o 
We will be following the HRP-090 consent process. 
Non-English Speaking Subjects 
• 
Non-English patients will be excluded from the current study 
due to inability to translate. 
Waiver or Alteration of Consent Process (consent will not be 
obtained, required information will not be disclosed, or the 
research involves deception) 
• 
N/A we will obtain consent. 
Subjects who are not yet adults (infants, children, teenagers) 
• 
N/A 

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disease patients 
 
 
Page 19 of 19 
Revised: December 1, 2018 
Adults Unable to Consent      N/A 
23.0 Process to Document Consent in Writing 
23.1 We are using the common consent form for patients. 
23.2 We will obtain written consent and then doctor’s approval of PAD 
patients. All healthy volunteers will not be required to get physician 
approval but will also document consent in writing.  
24.0 Setting 
24.1 Subjects with PAD will be identified and recruited from Vascular 
Surgery Associates Clinic. Screening visit for healthy volunteers will 
take place at FSU College of Medicine. Study procedures (for all 
subjects) for Visit 2 and 3 will be conducted at FSU College of 
Medicine facilities. 
• 
For research conducted outside of the organization and its 
affiliates describe: N/A 
25.0 Resources Available 
25.1 Describe the resources available to conduct the research: For 
example, as appropriate: 
• 
Given that the percent of individuals over 60 years of age with 
PAD is 12-20% we believe this allows us with a sufficient pool 
of individuals to recruit in the Tallahassee area. 
• 
80% of Matthew Martenson, 10% of Dr. Caldwell and 15-20% 
of Dr. Muller-Delp’s time will be committed to this study. 
• 
Our space for human research is approximately 1000 sq feet. 
We have access to patient rooms in the MRI facility where we 
are conducting part of the current study. This room is outfitted 
with chairs and an examination table we will use for resting 
measurements. We are currently using these facilities on 
another human study with splints and muscle stretching. Down 
from the MRI room we have access to a hallway where we will 
conduct the 6MWT. 
• 
We are located within the College of Medicine and will have 
an AED close by. In addition, we will have a College of 
Medicine physician (Dr. Lawrence D. Kaelin) that will have 
oversight of the study. He will not directly interact with 
patients; however, he is aware of the protocols being followed. 
• 
All individuals will be trained to understand data collection 
and methods prior to first patients with mock run throughs to 
ensure that all research runs accordingly and will be the 
primary individual taking measurements. 
26.0 Multi-Site Research*    --  N/A