Nicotinamide Riboside as an Enhancer of Exercise Therapy in Hypertensive Older Adults (The NEET Trial)
Phase: PHASE1
Conditions: Hypertension
Sponsor: University of Florida

 
 
 
 NCT04112043  
Nicotinamide riboside as an 
Enhancer of Exercise Therapy 
in hypertensive older adults  
- The NEET Trial  
PI: Robert T. Mankowski, PhD 
2-9-2023 
 

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Study Protocol and Statistical Analysis Plan 
1. Project Title: 
Nicotinamide riboside as an Enhancer of Exercise Therapy in hypertensive older adults – The NEET Trial. 
2. Investigators: 
Robert Mankowski, PhD 
Stephen Anton, PhD 
Christiaan Leeuwenburgh, PhD 
Demetra Christou, PhD 
Bhanuprasad Sandesara, MD 
Peihua Qiu, PhD 
3. Abstract:   
More than 80% of older adults have hypertension, with higher prevalence of high systolic blood 
pressure (SBP) putting them at high risk for cardiovascular (CV) disease and death. Because drug 
therapy that lowers SBP is associated with side effects such as hypotension, syncope, and kidney 
dysfunction, there is a great need for effective lifestyle SBP-lowering interventions for the older 
population that can replace drug therapy. While aerobic exercise is a recommended lifestyle 
intervention for controlling SBP and preventing CV disease naturally, in older adults it has been shown 
to be less effective in vascular-tissue remodeling because of arterial stiffness, resulting in less efficient 
SBP control. Reduced bioavailability of nicotinamide adenine dinucleotide (NAD+), a cofactor for the 
deacetylase sirtuin1 (SIRT1), may contribute to age-related vascular dysfunction via oxidative stress 
and reduced nitric oxide (NO). Exercise-induced overexpression of NAD+-dependent SIRT1 improves 
the bioavailability of NO. Preclinical evidence suggests that poor vascular-function improvement in 
response to exercise in older mice is caused by insufficient NAD+ levels to stimulate SIRT1 activity. 
Importantly, replenishment of NAD+ levels induced vascular remodeling, improved vascular function, 
and reduced SBP in mice. An objective of this study, therefore, is to test a combination of aerobic 
exercise and nicotinamide riboside, a compound that replenishes NAD+ levels, to optimize exercise’s 
SBP-lowering effect in hypertensive older adults. Initial human clinical trials demonstrated that 
nicotinamide riboside supplementation (1,000 mg/day) was safe and showed a higher potential to 
reduce SBP and arterial stiffness in participants with elevated SBP. As we have preclinical evidence 
that combining NAD+ replenishment with exercise is an ideal strategy for improving vascular function, 
our central hypothesis is that the intervention of aerobic-exercise training combined with nicotinamide 
riboside supplementation will reduce SBP in hypertensive older adults more effectively than will 
exercise alone. We will enroll 54 participants ≥ 55 years and older into either: (1) 1,000 mg/day of 
nicotinamide riboside plus 3 days/week of supervised, center-based walking exercise (n=18), or (2) the 
same exercise program combined with placebo (n=18), or (3) 1,000 mg/day of nicotinamide riboside 
alone (n=18). All participants will undergo daytime continuous SBP at baseline, 3 weeks, and 6 weeks, 
and arterial-stiffness measurements by pulse-wave velocity at baseline and at 6 weeks. Elevated SBP 
will be determined as daytime average equal to or above 130 mmHg, measured by the 24-hour blood-
pressure device. To our knowledge, this study will be the first attempt to enhance exercise therapy with 
nicotinamide riboside in hypertensive older adults. We believe that nicotinamide riboside is “the missing 
piece of the puzzle” in improving vascular remodeling and SBP management in older adults. 
Preliminary evidence from this pilot study may support a full-scale Phase III clinical trial in hypertensive 
older adults. The ultimate goal of this line of research is to find adjuvant strategies to improve the 
exercise’s SBP-lowering effects in older adults. 

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4. Background:  
More than 80% of older adults are diagnosed with hypertension and thus are at high risk of 
atherosclerosis, cardiovascular (CV) events, and death [1]. Systolic blood pressure (SBP) is the most 
common form of hypertension and is considered more important than diastolic blood pressure (BP) as 
an independent CV disease risk factor [2]. SBP equal to or above 130 mmHg needs treatment, as even 
10-mmHg elevation doubles the risk of a heart attack [3]. Because successful reduction of SBP with 
medication is often accompanied by side effects such as hypotension, syncope and kidney dysfunction 
[2], there is a great need for effective lifestyle SBP-lowering interventions in the older population. 
The major contributors to high SBP are decline in endothelial function, decline in capillary vessel 
density, and arterial stiffening, which are profound changes in vascular aging [4, 5]. Reduced 
bioavailability of nicotinamide adenine dinucleotide (NAD+), a cofactor for the deacetylase sirtuin1 
(SIRT1), may contribute to age-related vascular dysfunction via oxidative stress and reduced nitric 
oxide (NO) [6]. Endothelial dysfunction reduces bioavailability of NO, which increases arterial stiffness 
and elevates SBP [6]. Interventions that elevate NAD+ levels and increase SIRT1 activity may be 
critical for improving therapies that target endothelial dysfunction and arterial stiffness to manage SBP 
and prevent CV disease in older adults.  
While aerobic exercise is recommended for SBP control and CV disease prevention [7], it has been 
shown to be less effective in lowering SBP in older adults because of arterial stiffness [8]. Preclinical 
evidence suggests that age-related lower vascular function improvement in response to exercise may 
be caused by insufficient levels of NAD+ to stimulate SIRT1 activity and induce vascular remodeling for 
SBP reduction [9]. In particular, preclinical evidence showed that in old mice with replenished NAD+ by 
administration of nicotinamide mononucleotide (NMN), exercise was more effective in stimulating 
SIRT1 activity and restoring endothelial function and capillary density, compared to mice with lower 
NAD+ levels [9]. Such findings suggest that replenishment of NAD+ levels with metabolites (e.g., 
nicotinamide riboside (NR) or NMN) may be the key to enhancing vascular function in response to 
aerobic-exercise training and, thus, reducing SBP in older adults more effectively. 
In healthy, men and women aged 55 to 79 years, 6 weeks of supplementation with 1,000 mg/day of NR 
was well-tolerated and increased NAD+ levels in peripheral blood [10]. Importantly, NR 
supplementation produced trends towards reductions of SBP as well as arterial stiffness compared to 
placebo [10]. A post-hoc analysis of the same study sample showed trends towards larger reductions 
after NR supplementation in individuals with higher SBP (9 mmHg lower after NR compared to placebo 
among participants with 120–139 mmHg SBP) and arterial stiffness (41.5 centimeters per second 
(cm/s)) [10]. We believe that promising results from Martens et al., study and promising preclinical 
evidence from Das et al., that showed a combination of NAD+ replenishment and exercise was an 
optimal strategy to most effectively improve vascular function and reduce SBP in older mice, support 
our novel proposal that a combination of NR and aerobic exercise (NREx) will be more effective than 
either NR or exercise alone (exercise and placebo - PLEx) in hypertensive older adults. Thus, our 
central hypothesis is that the intervention of aerobic-exercise training combined with NR 
supplementation will reduce SBP in older adults who have high SBP more effectively than will exercise 
alone. 
To test our central hypothesis, we will recruit 54 older adults ≥55 years old with high 24-hour 
average SBP and follow them for 6 weeks in a double-blind fashion. Participants will be randomly 
assigned to receive either (1) 1,000 mg/day of NR plus 3 days/week of supervised, center-based 
walking exercise, or (2) the same exercise program combined with placebo, or (3) 1,000 mg/day of NR 
alone. Inclusion of all treatment groups will be important for detecting the potential benefit of NREx. 
High SBP will be determined at screening as daytime average above or equal to 130 mmHg and below 
160 mmHg, measured by the 24-hour BP device. All enrolled participants will undergo daytime 
continuous SBP measurements at baseline, 3 weeks, and 6 weeks, and arterial-stiffness 
measurements by pulse-wave velocity (PWV) at baseline and 6 weeks. 

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5. Specific Aims: 
The objective of this study is to conduct a pilot study to refine and finalize elements critical to 
conducting a future, definitive randomized, controlled trial (RCT). We propose to recruit 54 persons 
aged ≥ 55 years with SBP ≥130 mmHg and <160 mmHg for 6 weeks. Participants will be randomly 
assigned to receive either (1) 1,000 mg/day of NR plus 3 days/week of supervised, center-based 
walking exercise, or (2) the same exercise program combined with placebo, or (3) 1,000 mg/day of NR 
alone. Inclusion of all treatment groups will be important for detecting the potential benefit of NREx. 
High SBP will be determined at screening as daytime average above or equal 130 mmHg and below 
160 mmHg, measured by the 24-hour BP device. All enrolled participants will undergo daytime 
continuous SBP measurements at baseline, 3 weeks, and 6 weeks, and arterial-stiffness 
measurements by pulse-wave velocity (PWV) at baseline and 6 weeks. This study will enable us to 
address the following aims, all of which are of critical importance for designing a full-scale trial. 
Aim 1. To obtain critical data necessary to project the sample size needed for a full-scale trial 
using the change in SBP, as the primary outcome and; 
Aim 2. Using the change in arterial stiffness, as the secondary outcome. 
6. Research Plan: 
 
We propose to conduct a randomized, controlled pilot trial to test if nicotinamide riboside improves an 
effect of aerobic exercise training on cardiovascular function among hypertensive older adults. Following 
study entry, participants (N = 54) will be randomly assigned to either NREx, NR or PLEx groups for 6 
weeks. Exercise groups will be involved in three center-based walking training sessions per week. We 
anticipate that up to an additional 20 individuals enrolled may discontinue participation in the project prior 
to completion, and that we may have to screen up to 250 additional individuals to reach our recruitment 
goal of 54 subjects.  
 
6.1 Participants: Eligible participants will be males or females ≥ 55 years of age with daytime average 
SBP ≥ 130mmHg and < 160 mmHg. 
 
Table 3.1.1. Inclusion and exclusion criteria. 
Inclusion Criteria  
• 
Age 55 years and older   
• 
Daytime average of systolic blood pressure of ≥ 130 mmHg and < 160 mmHg.  
• 
Sedentary lifestyle, defined as < 150 min/wk of moderate physical activity as assessed by the CHAMPS 
questionnaire.  
• 
Willingness to be randomized to either treatment group 
• 
Willingness to participate in all study procedures 
Exclusion criteria  
• 
Failure to provide informed consent. 
• 
Pregnant 
• 
Change in blood pressure therapy (type or dose) within the last 3 months- Temporary Exclusion 
• 
Daytime average of systolic blood pressure ≥160 mmHg. 
• 
Regular consumption of nicotinamide riboside supplement 
• 
Current involvement in supervised rehabilitation program  

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• 
Absolute contraindication(s) to exercise training according to American College of Sports Medicine 
guidelines [11] 
• 
Daytime average of systolic blood pressure below 130mm Hg or Diastolic BP ≥ 100mm Hg. 
• 
Peripheral vascular disease; peripheral neuropathy; retinopathy 
• 
Severe cardiac disease, including NYHA Class III or IV congestive heart failure, clinically significant 
aortic stenosis, history of cardiac arrest, use of a cardiac defibrillator, or uncontrolled angina;   
• 
Myocardial infarction or stroke within past year 
• 
Significant cognitive impairment, including known dementia diagnosis or a Mini-Mental State Examination 
exam score < 24  
• 
Progressive, degenerative neurologic disease, e.g., Parkinson’s Disease, multiple sclerosis; 
• 
Severe rheumatologic or orthopedic diseases, e.g., awaiting joint replacement, active inflammatory 
disease; 
• 
Severe pulmonary disease, requiring steroid therapy or the use of supplemental oxygen; 
• 
Hip fracture, hip or knee replacement, or spinal surgery within past 4 months; 
• 
Other significant co-morbid conditions that would impair ability to participate in the exercise-based 
intervention 
• 
Simultaneous participation in another intervention trial 
 
6.2 Recruitment: Participant recruitment will be coordinated in conjunction with the OAIC Clinical 
Research Core, led by co-investigator Dr. Stephen Anton. We will develop a targeted recruitment 
approach based on previous experiences utilizing methods of recruitment that include direct mailings, 
newspaper classified and print ads, community luncheons and health fairs, as well as clinic referrals. We 
have been extremely successful in implementing this approach in past studies 
 
6.3 Screening and Study Entry: Participants deemed eligible based on an initial phone screening by our 
front desk manager, will be encouraged to consult with their physician and invited to an in-person 
screening visit. During this visit, potential participants will first be asked to give their informed consent 
and then will be screened for study entry criteria. Initial screening procedures will include a review of 
their medical history, medication use, a physical exam or nursing assessment, physical activity levels, 
cognitive function (assessed by the Mini-Mental State Exam), measurement of vital signs, height and 
weight, BMI (calculated in database only), collection of demographics information, single 24-hour 
ambulatory SBP monitoring (daytime average SBP≥130 mmHg and <160 mmHg – qualifying criterion), 
and urine collection for a pregnancy test. If all study entry criteria are met, participants will be scheduled 
to return to the clinic for baseline assessments. In addition, the study participants will be asked to sign 
Medical Records Release Authorization if they qualify. This Authorization will be used exclusively to 
obtain medical records to acquire facts, details, and outcomes of Serious Adverse Events that occurred 
during the study and have to be reported to the sponsor and/or IRB. 
 
6.4 Baseline Assessment and Randomization: If all study entry criteria are met, participants will be 
scheduled to return to the clinic for baseline assessments prior to randomization. Participants who have 
not yet had a physical evaluation or nursing assessment will complete this before undergoing physical 
tests. Participants will be asked to provide a fasting blood sample for evaluation of clinical safety lab 
values. We will also collect urine samples (first sample collected between 8 am to 11 am and another 
sample collected between 5 pm to 8 pm) at this visit, which will be slow frozen in a -80 freezer and 
analyzed after data collection is complete. The first sample will be collected when the participant comes 
in the morning to participate in the in-person visit. Then, the study coordinator will provide the urine 
sample collection cup for the participant to bring back home to collect the second urine sample. The 
study coordinators can help transport the second urine sample or, if the participant prefer, the 
participant could bring the urine sample back to the study coordinator. In addition, vital signs and weight 

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will be measured, as well as exercise capacity with the 6-Minute Walk Test and self-assessed 
functionality with the Late Life Function and Disability Instrument (LLFDI). Participants will also be given 
the 24-h BP device to complete the 24-hour ambulatory recording and will have their arterial stiffness 
assessed. We will also collect adverse events and update medication changes. Finally, the study 
coordinator will provide participants with a supplement intake form to take home to record when the 
supplement is taken. Staff members will then discuss randomization procedures with participants. The 
participants will receive a return envelope/box to return the ambulatory BP device after the 24-hour 
recording. 
 
6.5 Exercise Intervention: After random assignment to either the NREx or the PLEx groups, participants 
will engage in a three-days a week, center-based walking exercise intervention for 6 weeks. Following a 
brief warm-up, participants will be instructed to walk for 30 minutes at a 5-6 on the CR10 scale with 
encouragement for 10 minutes to be vigorous (7-8 on CR10 scale). Sessions will also include balance 
training to promote cool-down. Participants will be introduced to exercises in such a way that they begin 
with lighter intensity and gradually increase. The progressive nature of the intervention is designed to 
minimize discomfort and prevent injury. Blood pressure will be measured prior to each exercise 
session; the participant will not complete the session that day if he or she has an in-person SBP >200 
mmHg or DBP >110 mmHg (AHA guidelines for exercise contraindication) measured at the exercise 
center. 
 
6.6. Supplementation: Participants will be randomly assigned to receive, in a double-blind fashion, 
either 1,000 mg/day of encapsulated NR (Niagen®) or placebo. The company ChromaDex (Irvine, CA) 
will provide both the NR and placebo capsules for the proposed study. Placebo capsules will be 
identical to those including NR. As noted above, a 6-week NR study of Martens et al. demonstrated that 
NR supplementation at a dose of 1,000 mg/day was safe in rather healthy middle-aged and older 
adults. Subjects will ingest NR (NIAGEN®; 500 mg, twice per day; ChromaDex, Inc.) or alike looking 
placebo capsules [10]. The participants will be provided with capsules sufficient for at least 3 weeks. 
The second 3-week portion of the capsules will be dispensed at 3-week follow-up visits by a study 
coordinator. A study coordinator will be given a jar with the study product by an unblinded staff person 
not-involved in this study. The not-involved staff member will be is possession of the identified 
randomization list and will provide this list after completing the enrollment and outcome measure 
analyses. The participants will receive a supplement intake diary to note the taken pills and a study 
coordinator will count the remaining pills at 3 and 6-week visits. 
 
Data from the exercise interventions will be collected and stored in the study database. Following each 
session, participants will be asked to provide a rating of perceived exertion (RPE) for the session 
according to the Borg CR10 scale.  
Table 1. Data collection summary by study visit. 
Study phase 
Pre-randomization 
Randomization 
Visit description (FU=follow-up, CO=close-out) 
Screen 
Baseline 
FU 
CO 
Visit number 
1 
2 
3 
4 
Week number 
-2 
0 
3 
(+ or - 7 days) 
6 
(+ or - 7 days) 
Informed consent, review of inclusion/exclusion 
criteria 
X 
 
 
 
Medical history and medications, MSSE 
X 
 
 
 
Height 
X 
 
 
 
Weight 
X 
X 
X 
X 
BMI (calculated in database only) 
X 
 
 
 
Demographics information 
X 
 
 
 
Physical Exam/nursing assessment 
X* 
X* 
 
 
Monitor vital signs 
X 
X 
X 
X 
Ambulatory BP recording 
X 
X 
X 
X 

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6.7 
Adherence to interventions. To enhance adherence to the study interventions (compliance), we will 
utilize empirically-supported techniques which we have successfully utilized in prior studies. Our first 
step will be to fully inform participants of study requirements before randomization and enroll only those 
persons who are willing to complete all study procedures. Undoubtedly, however, issues arise after 
randomization with the potential to limit compliance. Common causes of poor compliance include 
vacations, spouse care, fatigue, and fluctuations in motivation, perceived lack of benefit, physical 
discomfort, and adverse health experiences. First, we have designed the trial, including the 
inclusion/exclusion criteria and physician monitoring, to limit the likelihood of experiencing adverse 
health events in response to the interventions. In addition, care will be taken by the interventionists to 
promote the safe engagement in the exercise intervention. For other issues of non-compliance, we will 
approach this potential problem using a social problem solving model (“toolbox”) approach in dealing 
with individual adherence problems.28 This approach assumes that the process of changing behavior is 
a collaborative effort between the participant and the interventionists, and that behavior change can be 
readily undertaken during the regular weekly contacts that intervention staff will initiate throughout the 
course of the intervention, as well as during face-to-face visits. Our team has successfully applied this 
type of problem-solving approach in promoting adherence to behavioral interventions in prior studies. 
 
6.8 Follow-up and Close-out visits. In addition to baseline and screening visits, assessment visits will be 
conducted at approximately 3 weeks (follow-up) and approximately 6 weeks post-randomization. During 
the 3-week assessment visit, the research team will evaluate participant’s vital signs, measure weight, 
update medical history, collect blood samples for safety markers and inquire about any adverse 
experiences since the last visit. We will also collect urine samples (first sample collected between 8 am 
to 11 am and another sample collected between 5 pm to 8 pm) at these visits, to be analyzed after data 
collection is complete. The first sample will be collected when the participant comes in the morning to 
participate in the in-person visit. Then, the study coordinator will provide the urine sample collection cup 
for the participant to bring back home to collect the second urine sample. The study coordinators can 
help transport the second urine sample or, if the participant prefer, the participant could bring the urine 
sample back to the study coordinator. Blood pressure will be measured prior to completing physical 
function tasks at these visits; the participant will not complete the physical function tasks for that visit if 
he or she has an in-person SBP >200 mmHg or DBP >110 mmHg (AHA guidelines for exercise 
contraindication) at the clinic. The coordinator will collect and count return pills and dispense a new 
supply. At the end of the 3 week visit, the participant will be provided with a 24-Hour Ambulatory Blood 
Pressure Monitor and the measurements will be recorded as in previous visits. During the close-out (6-
week) visit, the team will perform the same evaluations as during the baseline visit, as well as collect 
and count returned pills, and collect supplement intake information from participants. We will also 
collect adverse events and update medication changes. 
 
CHAMPS Questionnaire 
X 
 
 
 
Pregnancy test 
X 
 
 
 
6 minute walk test 
 
X 
 
X 
Late Life Function & Disability Instrument 
 
X 
 
X 
Randomization 
 
X 
 
 
Blood collection 
 
X 
X 
X 
Urine collection 
 
X 
X 
X 
Arterial stiffness 
 
X 
 
X 
 Dispense Supplement Intake Diary 
 
X 
X 
 
Dispense Study Supplement (NR or Placebo) 
 
X 
X 
 
Pill counts, collect diaries, assessment of 
adverse experiences 
 
 
X 
X 
Collection of AEs and update medication 
changes 
 
X 
X 
X 
*The physical exam/nursing assessment can be completed at either Screening Visit 1 or at the Baseline Visit as long as it is 
prior to randomization. 
 

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6.9. Primary and Secondary Outcome Measures.  
 
6.10 Sample size justification. This pilot study will help generate precise estimates of effect variance in 
the population in which a subsequent larger-scale study will be conducted. Published recommendations 
for the design of pilot studies indicate that a sample of 18 participants per randomized arm is typically 
sufficient to estimate a parameter for a future trial [14-16]. The enrollment sample of 54 (n=18 x 3 arms) 
participants is intended to provide preliminary data necessary to indicate feasibility of a larger trial and 
to provide descriptive estimates of effects. Based on our experience from the past and current clinical 
trials conducted at our institute, we do not expect an attrition higher than 15%, therefore, we increased 
a group size to n=18, which will give us sufficient sample sizes for the pilot purposes after drop-out 
(n=15 x 3 groups). The data will also provide for nominal estimation (using a 95% CI) of mean changes 
in dependent variables within each arm. By assuming the mean decrease in SBP after a 6-week of 
intervention to be 20 mmHg, 10 mmHg and 15 mmHg in the three groups of NREx, PLEx and NR, 
respectively, and the standard deviation of the SBP decreases to be 10 [16], the sample size of 15 
participants for each group can guarantee a power of 85% to detect the difference between the NREx 
and PLEx groups and a power of about 80% to detect the differences among the three groups. We 
expect that the differences of the arterial-stiffness measurements among the three groups are larger 
than those of SBP. Thus, the power numbers for that measure will be larger as well.  
 
6.11 Safety monitoring plan. To ensure participant safety in the proposed study, we will adapt procedures 
that we have implemented successfully in other studies including LIFE,46,47 Weight Loss + Exercise,48 and 
Task Specific Exercise.49 As he has for a number of our prior studies, Dr. Bhanuprasad Sandesara will 
serve as the Study Physician who is responsible for assessment, monitoring and interventions for health 
events. To maximize participant safety we will follow a standardized screening protocol. Accordingly, all 
potential participants will undergo screening for cardiovascular and other major diseases by means of a 
health questionnaire, medication inventory, and physical exam/nursing assessment. Those with 
conditions that meet the exclusion criteria described in section 6.1 as determined by Dr. Sandesara will 
be excluded. Dr. Sandesara will also review clinically significant abnormal laboratory values and indicate 
the need for study withdrawal if he deems it necessary. Each participant will be instructed to report the 
occurrence of an adverse event at scheduled data collection times (scheduled clinical exams or phone 
interviews). Participants also have access to study clinic personnel at other times to report serious 
adverse events or concerns about the safety of participating in the study. If necessary, Release of 
Medical Records Authorizations signed by the study participants will be used to obtain medical records to 
collect details and outcomes of SAEs for safety and reporting purposes. 
Venipuncture will be performed by a trained and experienced phlebotomist using standard techniques. 
Participants will always have venipuncture performed while seated upright with both feet on the floor.  
24-h BP 
All blood pressure recordings will be performed with validated Oscar 2 monitors, under 
the supervision of trained personnel and according to the manufacturer’s 
recommendations. SBP will be measured every 20 minutes (min) during the day (from 
0700 to 2200 hours), and every 60 min overnight (from 2200 to 0700 hours) [12]. 
Arterial 
Stiffness 
 
Aortic pulse-wave velocity (PWV), commonly measured as carotid to femoral PWV 
(cfPWV), is considered to be the gold standard method of assessing arterial stiffness 
in humans. cfPWV will be measured using the SphygmoCor XCEL system (AtCor 
Medical, New South Wales, Australia). Briefly, cfPWV will be determined by recording 
pressure pulse waves at the carotid and femoral arteries using a high-fidelity 
micromanometer (Millar Instruments, Houston, TX) and calculating the distance 
between the recording sites divided by the time delay between the carotid and the 
femoral pulse waves [13]. 

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Center-based assessments and interventions will be conducted and supervised by trained staff who will 
monitor potential adverse experiences and symptoms. All assessors and interventionists receive CPR 
training and training on management of acute events including syncope, chest pain, acute dyspnea, focal 
neurological symptoms and abnormal vital signs. Portable defibrillators are available at each intervention 
and assessment site and all study staff have on-call access to the study physician and contact numbers 
for emergency services. Institutional and community EMS services will be activated if needed. As 
indicated previously, participants will be taught the importance and proper method of warming-up prior to 
and cooling-down following structured activity sessions. If at any point during an exercise session, 
participants develop chest pain, shortness of breath, or dizziness, they will be instructed to rest and to 
contact the center and their physicians if these symptoms persist or recur with further exercise.  
Blood pressure and heart rate will be measured before and after the resistance training at each center 
based session for pre-exercise safety. The participant will not complete the session that day if he or she 
has an in-person SBP >200 mmHg or DBP >110 mmHg (AHA guidelines for exercise contraindication) 
measured at the exercise center.  
 
Procedures to minimize discomfort include warm-up and cool-down activities. The participants will also be 
introduced to the intervention exercises in a structured way, such that they begin with lighter intensity and 
gradually increase over the course of the first week. During the intervention visits, participants will be 
supervised at all times. Participants will also be instructed to move slowly when rising from a seated or 
lying position to reduce the risk of experiencing orthostatic hypotension. Study staff will be instructed to 
stand in a supportive position when participants are rising to prevent a fall in case of syncope. Exercise 
will be stopped if the participant reports pain, tightness or pressure in the chest, significant shortness of 
breath, feeling faint, lightheaded or dizzy, or significant other medical problems.  
Any significant adverse events will be reported promptly to the IRB and to the NIA Data Safety 
Monitoring Board (DSMB). Otherwise, annual reports will be prepared for both.   
Written informed consent will be obtained after explanation to subjects of all procedures and time 
commitments. The study interviewers will explain to prospective participants the purpose, methods and 
extent of the study. Potential participants will be asked to read the informed consent form and to ask 
questions. The form will be written in simple, easy-to-understand language. Staff members will also 
review all key aspects of the study verbally.  
Confidentiality of data will be maintained by using research identification numbers that uniquely identify 
each individual. Data will be used only in aggregate and no identifying characteristics of individuals will 
be published or presented. Safeguards will be established to ensure the security and privacy of 
participants’ study records. The information collected from participants in this study has a low potential 
for abuse, since the data do not address sensitive issues. Nevertheless, appropriate measures will be 
taken to prevent unauthorized use of study information. The research records will be kept in a locked 
room at the Institute on Aging. The files matching participants' names and demographic information 
with research ID numbers will be kept in a separate room and will be stored in a locked file that uses a 
different key from that of all other files. Only study personnel will have access to these files, and they 
will be asked to sign a document that they agree to maintain the confidentiality of the information. 
After the study is completed, local data will be stored with other completed research studies in a 
secured storage vault. In compliance with the Health Insurance Portability and Accountability Act 
(HIPAA) and the Standards for Privacy of Individually Identifiable Health Information of the Department 
of Health and Human Services, we will access personal health information and medical records only 
after receiving signed informed consent, as described above.  
Finally, the study protocol will be registered at www.ClinicalTrials.gov before study enrollment begins. 
7. Possible Discomforts and Risks: 

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The first clinical trial with a 6-week NR supplementation (1000 mg/day) in middle-aged and older adults 
showed that adverse events reported over the course of the study did not differ between the treatment 
group and the placebo. We will test the same supplementation dose and duration provided to us by the 
same company. A different study that tested an effects of a different product but containing NR (500 or 
1000mg/day) and Pterostilbene showed no serious adverse effects among older adults other than 
nausea, fatigue, headaches, diarrhea, stomach discomfort and indigestion. We will inform the potential 
participants that supplementation with similar products containing NR in older adults was associated with 
these side effects and there is always a risk that they may experience these side effects. 
Potential risks are those associated with health information privacy, blood pressure testing, venipuncture 
and participation in exercise training and testing.  
The pregnancy test may provide a false-positive or false-negative result. 
Venipuncture can be associated with pain, bruising, hematoma formation, superficial phlebitis, and rarely 
cellulitis or fainting. The risks of placing a blood pressure cuff on a participant are that it may cause 
pinching, slight bruising, discomfort, or skin irritation from the cuff. The primary risk associated with 
moderate-intensity exercise training is skeletal muscle soreness. There are also other risks that relate to 
falls and fractures, exacerbation of arthritis and other joint conditions, post-exercise hypotension, and 
cardiovascular events. There is a risk that a participant may trip, stumble, or fall during the exercise 
sessions and experience shortness of breath, dizziness, palpitations, chest pain or discomfort, heartburn, 
light headedness, or feeling about to faint. Exercise is contraindicated when resting SBP is >200 mmHg 
or DBP > 110 mmHg. We are recruiting participants with systolic hypertension of ≥ 130mmHg and < 160 
mmHg, therefore, there is no specific increased risk to these participants over the general risks 
associated with moderate-intensity exercise training.  
The DSMB will be set-up and run by the National Institute on Aging.  
Confidentiality:  Data will be used only in aggregate and no identifying characteristics of individuals will 
be published or presented. Confidentiality of data will be maintained by using research identification 
numbers that uniquely identify each individual. Safeguards will be established to ensure the security 
and privacy of participants’ study records. Appropriate measures will be taken to prevent unauthorized 
use of study information. Data other than demographic information will not use names as an identifier. 
The research ID number will be used. The research records will be kept in a locked room in the study 
site. Only trained and certified study personnel will have access to these files, and they will be asked to 
sign a document that they agree to maintain the confidentiality of the information.  Electronic records 
will be stored on a password protected network server maintained by the university information 
technology department.  In compliance with the Health Insurance Portability and Accountability Act 
(HIPAA) and the Standards for Privacy of Individually Identifiable Health Information of the Department 
of Health and Human Services, we access personal health information and medical records only after 
receiving signed informed consent. If a participant does not qualify based on the initial phone pre-
screening, we will delete the individual’s identifying information from the study record immediately, but 
we will keep the other information provided to track the outcome of our calls. 
8. Possible Benefits: 
Importantly, the proposed project should have tangible benefits for participants. These benefits include 
information about their health and assessments of their cardiovascular status. All study participants will be 
encouraged to communicate the results from the study to their primary care providers. Moreover, 
participants in exercise groups will receive supervised exercise training and instruction about how to 
maintain exercise habits at home. Participants in the NR alone group may experience potential benefits of 
the supplementation. We expect that these benefits will improve quality of life for all participants. 

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Potential benefits of NR alone may be lowering systolic blood pressure and improving vascular flexibility, 
but the combination of NR and exercise may have better effects than exercise or NR alone on systolic 
blood pressure reduction and improvement of vascular flexibility in hypertensive older adults. 
9. Conflict of Interest: 
None 
10. Statistical Analysis Plan 
The primary analysis will follow an “intent-to-treat” model. Because repeated measures are obtained on 
individual participants and they are potentially correlated, a mixed-effect linear regression model will be 
used. Differences in outcome measures at the 3-week follow-up (or 6-week close-out) from baseline for 
each individual participant will be used as responses, and two dummy variables representing the three 
treatment groups, age, and gender, and other baseline characteristics of participants will used as 
covariates. A random-effect term will also be included in the model to accommodate within-subject data 
correlation. This analysis will be performed for each outcome variable. The relatively small sample size 
may imbalance pre-randomization covariates. Thus, caution will be taken in the interpretation of 
hypothesis testing results. 
 

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