Dose-response Effect of Dietary Nitrate on Muscle Function in Older Individuals
Phase: PHASE1
Conditions: Aging, Sarcopenia
Sponsor: Indiana University

 
 
 
 
 
 
 
 
Protocol Document 
 
Dose-response Effect of Dietary Nitrate on Muscle Function in Older Individuals 
 
NCT03595774 
 
June 3, 2019 

Protocol Version 03June2019 
 
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Dose-response effect of dietary nitrate on muscle 
function in older individuals 
 
 
Principal Investigator: 
Andrew Coggan, Ph.D. 
Associate Professor, Department of Kinesiology 
I.U. School of Health and Human Sciences 
 
 
 
 
 
 
Sub-Investigators:  
Ranjani N. Moorthi, M.D. 
 
 
 
 
Sharon Moe, M.D. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol Version 03June2019 
 
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Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Inclusion/Exclusion Criteria 
4.0 
Enrollment/Randomization 
5.0 
Study Procedures 
6.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
7.0 
Study Withdrawal/Discontinuation 
8.0 
Statistical Considerations 
9.0 
Privacy/Confidentiality Issues 
10.0 Follow-up and Record Retention 
11.0 References 
12.0 Appendix  
 
 

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1.0 
Background 
By 2050, approximately 20% of the US population, or >80 million people, will be 65 y of age or 
older.1 Unfortunately, aging is accompanied by a progressive reduction in the maximal strength, 
speed, and especially power of skeletal muscle. These age-related physiological changes often 
lead to functional limitations that are highly predictive of disability, institutionalization, and 
mortality in the elderly.2,3 Thus, any intervention that significantly enhances muscle contractile 
function could potentially improve the health, quality of life, and possibly even the longevity of 
older individuals. 
Numerous factors undoubtedly account for the decline in muscle function described above, with 
age-related changes in the size, properties, and neural control of muscle likely all playing a role.4 
Another important factor, however, may be a fall in NO bioavailability with aging. Although initially 
identified as a vasodilator, i.e., as “endothelium-derived relaxing factor”, NO is in fact a key 
cellular signaling molecule with pleiotropic effects in many tissues. These include skeletal muscle, 
wherein among other effects NO helps modulate contractile function.5-9 With aging, however, 
whole-body NO production decreases, as evidenced, e.g., by a progressive decline in the plasma 
concentrations of its downstream metabolites, NO2- and NO3-.10,11 In skeletal muscle itself, there 
is a dramatic decrease in interstitial NO2- and NO3- concentrations,12 as well as a decline in the 
activity of the neuronal form of NO synthase (nNOS, or NOS1),13,14 the primary isoenzyme within 
muscle responsible for the synthesis of NO from L-arginine, O2, and NADPH. These changes are 
accompanied by an age-related reduction in flow-mediated vasodilation,15-18 perhaps the hallmark 
indicator of NO bioavailability. In turn, the latter (i.e., flow-mediated vasodilation) has been shown 
to correlate positively with muscular power and physical functioning in older men and women.19 
Taken together, these data suggest that decreased NO production may contribute to the age-
associated decline in muscle contractile properties and hence in functional capacity.  
In this context, the physiological effects of dietary NO3- are of considerable interest. It is now 
recognized that, rather than being metabolically inert or, worse, a potential carcinogen, NO3- in 
the diet is a significant source of NO in the body20-24 (Fig. 1). In fact, this dietary pathway, which 
entails the reduction of NO3- to NO2- by facultative anaerobic bacteria in the mouth followed by 
further reduction of NO2- to NO by, e.g., deoxyhemoglobin, can account for as much as ~25% of 
basal whole-body NO production.25,26 This dietary pathway serves as an important “backup” 
system to the more well-known NOS pathway. This is likely to be especially true in skeletal muscle, 
since unlike the NOS pathway the 
dietary pathway operates well at low 
pO2 and is stimulated rather than 
inhibited by low pH,20-24 conditions 
that regularly exist in muscle, both at 
rest and especially during contractile 
activity. Indeed, skeletal muscle has 
recently been shown to play a central 
role in whole-body NO3-/NO2-/NO 
metabolism.27,28 
Based on the above, it is logical to 
hypothesize that increased dietary 
NO3- 
intake 
might 
enhance 
NO 
bioavailability in older men and 
women, leading to an improvement in 

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muscle function. Indeed, we have observed significant increases in muscle speed and power 
following dietary NO3- intake in healthy younger subjects,29 athletes,30 and especially middle-aged 
heart failure patients.31 (See Preliminary Data.) Along the same lines, Haider and Folland32 and 
Whitfield et al.33 have reported that dietary NO3- intake enhances the rate of force development 
and peak force output of the quadriceps muscle of healthy young men during electrically-
stimulated contractions. To date, however, no study has specifically examined the impact of 
dietary NO3- on muscle contractile function in older persons. Furthermore, it cannot be 
automatically assumed that dietary NO3- will be equally efficacious in older subjects. For example, 
in rodents the effects of dietary NO3- are more prominent in fast- vs. slow-twitch muscle,34,35 and 
some,36-39 but not all,40-42 studies of humans have found that aging results in a reduction in the 
percentage of fast-twitch muscle fibers. Alternatively and/or in addition, more rapid destruction 
of NO due to increased production of reactive oxygen species by aging muscle43 could limit the 
beneficial effects of dietary NO3- in older persons. 
Even if the efficacy of a particular dose of NO3- could be established in older subjects, this would 
not necessarily mean that it is the optimal dose. There is limited data in the literature regarding 
the dose-response relationship for dietary NO3- for various outcomes, and only one study has 
examined the response to exercise over a wide range of doses.44 These authors found that acute 
ingestion of either 8.4 or 16.8, but not 4.2, mmol of NO3- improved performance equally during 
high intensity aerobic exercise in healthy young men. However, it is not known whether a similar 
plateau exists with respect to improvements in muscle contractile function, nor is it known 
whether aging influences this relationship. Such information is obviously critical to any attempt 
to pursue funding for larger (e.g., multicenter) trials evaluating dietary NO3- as a potential 
treatment for reduced muscle function in older persons, especially since the incidence of negative 
side effects (e.g., GI distress) may increase at higher doses.  
Our Preliminary Data (see below) suggest that older subjects may require a greater dose of NO3- 
than younger individuals to yield a significant improvement in muscle speed and/or power. 
Preliminary data 
Using methods essentially identical to those to be employed in the proposed experiments (see 
below), we previously demonstrated that acute dietary NO3- intake increased maximal knee 
extensor speed (Vmax) and power (Pmax) in 12 healthy, younger men and women by 11±5% 
(P<0.05) and 6±3% (P<0.05), respectively.29 We observed similar dietary NO3--induced 
improvements in muscle speed and power in 13 athletes performing multi-joint, multi-muscle, 
i.e., sprint cycling, exercise.30 Lastly, we found even greater increases in Vmax and Pmax (of 
12±5%, P=0.09, and 13±4%, P<0.05, y) in nine middle-aged patients with systolic heart failure 
(HF).31 
Using the same methods, we have also recently determined the effects of dietary NO3- in six 
healthy men and women 64-79 y of age. As shown in Table 1, plasma NO3- and NO2- and breath 
NO increased (P<0.05) by 1309±188%, 268±91%, and 142±19%, respectively, by 2 h after 
ingestion, and all three remained elevated for the remainder of the experiment. These findings 
are consistent with our previous research.29-31 This increase in NO bioavailability resulted in a 
9±5% increase in maximal muscle speed, i.e., Vmax (Fig. 2, left panel), with five out of the six 
subjects demonstrating an improvement. This increase, however, was not statistically significant 
(i.e., P=0.13), and maximal muscle power, i.e., Pmax, improved on average by only 2±3% 
 
 
 

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 (P=0.47) (Fig. 2, center panel). This is only 1/3rd as much as we previously found in younger 
men and women.29,30 Closer examination of the data suggested that this diminished effect was 
due to an inadequate dose of NO3-, as there was a significant positive relationship between NO3- 
intake relative to body mass and the relative improvement in Pmax (Fig. 2, right panel). These 
preliminary results therefore suggest that older individuals remain responsive to NO3-, but that a 
higher dose may be required to elicit improvements in muscle contractile function. 
2.0 
Rationale and Specific Aims 
In light of the above, we will use a double-blind, placebo-controlled, crossover design to address 
the following Specific Aim: 
Specific Aim #1:  
To determine the dose-response relationship between acute dietary 
NO3- supplementation and muscle contractile function in healthy, 
older (i.e., 65-79 y old) men and women. 
3.0 
Inclusion/Exclusion Criteria 
Inclusion:  
• 
Men and women age 65-79 
• 
In good health, as determined by the physician’s  review of history, physical examination, 
resting EKG, and routine blood tests  
Exclusion: 
• 
Men and women <65 or >79 years of age 
• 
Unable to provide informed consent 
• 
Currently pregnant or lactating 
Table 1. Changes in plasma NO3- and NO2- and breath NO in response to dietary NO3- intake in 
older study participant.  
 
Trial 
Pre 
1 h 
2 h 
10 min post 
exercise 
Plasma NO3- 
(μmol/L) 
Placebo 
26±4 
24±3 
24±3 
22±4 
Nitrate 
31±7 
285±27* 
295±27* 
282±33* 
Plasma NO2- 
(μmol/L) 
Placebo 
0.21±0.03 
0.26±0.06 
0.26±0.06 
0.20±0.03 
Nitrate 
0.27±0.04 
0.43±0.13 
  0.49±0.06* 
  0.56±0.10* 
Breath NO 
(ppb) 
Placebo 
23±5 
26±4 
27±4 
23±5 
Nitrate 
27±4 
  49±7* 
  38±4* 
  52±10* 
Values are mean±S.E. for n=7. *Nitrate trial significantly higher than Placebo trial at same time 
point (P<0.05).  
Figure 2. Responses of Vmax (left panel) and Pmax (center and right panels) to dietary NO3- in older subjects. 

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• 
Current smokers 
• 
Significant orthopedic limitations or other contraindications to strenuous exercise 
• 
Those taking phosphodiesterase inhibitors (e.g., Viagra) 
• 
Those taking proton pump inhibitors, antacids, xanthine oxidase inhibitors, or on hormone 
replacement therapy 
• 
History of major metabolic disease (e.g., type I and type II diabetes, thyroid disorders), 
history of neuromuscular disease (e.g., cervical spondylotic radiculomyelpathy, lumbar 
spondylosis, amyotrophic lateral sclerosis, Guillain-Barré syndrome, and acquired 
demyelinating polyneuropathies), cardiovascular disease (e.g., > stage II hypertension, 
heart failure, myocardial infarction/ischemia, significant myocardial or pericardial diseases 
(e.g. amyloidosis, constriction), moderate or severe valvular disease, renal disease, liver 
disease, or anemia 
Twenty  study participants will be enrolled. 
4.0 
Enrollment/Randomization 
Subjects will be recruited from the community through approved methods including flyers, emails 
from the Indiana Clinical and Translational Sciences Institute (All IN for Health), and being 
included in the searchable “All IN for Health” clinical trials database .   When potential study 
subjects contact the study team expressing an interest in the study they will be given additional 
information regarding the study procedures, requirements, and risks.  If they are still interested 
the study team will complete a brief phone screening to see if they qualify for an in-person 
screening visit.  As previously approved by amendment, potential subjects will be asked if they 
are part of the IU Health medical system and if so, do they give verbal permission for their records 
to be reviewed for possible exclusions to participation.  If they are not part of the IU Health 
system or do not give permission, they will still be able to participate in a screening visit if it 
appears they meet other pre-screening criteria.  If it appears they are eligible for a screening 
visit, they will be scheduled and a consent form will be mailed / emailed to them ahead of the 
screening visit for review.  After the study participant has had several days to review the consent 
form, a study team member will call to answer any questions the participant may have regarding 
the consent form. When the subject arrives for the screening visit a member of the research team 
will thoroughly review the consent with the study participant and answer questions. Study 
participants will be given as much time as they wish to consider participation before signing the 
consent form. If the study participant agrees to move forward with the screening visit, the consent 
will be signed by the study participant and the person who reviewed the consent and obtained 
the participant’s signature. A copy of the signed consent form will be provided. Both the 
participants and the investigators will be blinded to the order of treatment, which will be 
randomized using randomization.com. 
5.0 
Study Procedures 
Study Design 
Each subject will be studied using a double-blind, placebo-controlled, crossover design. Figure 3 
illustrates the flow through the study for an individual participant. 
 
Figure 3. Study design. 

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Study Procedures 
Participants will be asked to fast for 12 hours prior to each study day. 
On Study Day 1 (Pre-test), all study participants will complete a screening / phenotyping 
examination. The participant will undergo a complete history, physical exam, resting EKG and 
phlebotomy for screening and phenotyping laboratories (complete blood count, liver and kidney 
function tests, electrolytes, fasting glucose and insulin). They will also practice the entire isokinetic 
dynamometer exercise test.  
To minimize variation in baseline NO3-/NO2-/NO levels, participants will be instructed to consume 
their normal diet (aside from the BRJ supplement) throughout the study. Participants will, 
however, be asked to avoid consumption of high NO3- foods (e.g., beets, spinach, collard greens) 
the evening prior to testing. This approach is justified based on the short half-life of NO3- in 
plasma (i.e., ~8 h) as well as previous research demonstrating that even a chronic increase in 
dietary NO3- intake up to 2.5 mmol/d (i.e., ~3x normal dietary NO3- intake in the US) has no 
significant influence.56 Participants will be asked to refrain from chewing gum, alcohol, and 
caffeine-containing food/drinks for 24 h prior to each remaining visit. Participants will also be 
instructed to refrain from use of an antibacterial mouthwash throughout the study, since this 
would limit conversion of NO3- to NO2- by bacteria in the oral cavity.57 Participants will be asked 
to fast for 12 h prior to each remaining study visit. 
On Study Day 2, participants will undergo phlebotomy for plasma NO3- and NO2- measurement, 
and have their breath NO level determined. (Breath NO will be measured as a biomarker of 
increases in whole-body NO production in response to dietary NO3- intake.52,53) The subject will 
then ingest 3.3 mL/kg of a commercial concentrated beetroot juice (BRJ) supplement (Beet It 
Sport®, James White Drinks, Ipswich, UK) either: 1) essentially devoid of NO3- (placebo); 2) 
containing (as determined by previous direct measurement) 91 μmol/mL NO3- (thus total dose = 
300 μmol/kg, or 21 mmol in a 70 kg subject); or 3) an equal mixture of the placebo and NO3--
containing BRJ products. The placebo, which is prepared the company by extracting NO3- from 
BRJ using an ion exchange resin, is indistinguishable in packaging, color, taste, texture, and smell 
from the standard product, and does not alter plasma NO3-/NO2- or breath NO concentrations or 
the physiological responses to exercise.29-31 Additional blood and breath samples will be obtained 
1 and 2 h later, as well as blood pressure and heart rate, afterwhich time the subject will have 
the Vmax and Pmax of the knee extensor muscles of their dominant leg determined via isokinetic 
dynamometry (see below). The timing of all measurements has been chosen to correspond to 
peak plasma NO3-/NO2- concentrations and breath NO levels, which reach a maximum 2-3 h after 
ingestion.29-31 
On Study Days 3 and 4, participants will be asked to return to undergo the same studies as 
described above for Study Day 2. 
Study Methods 

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Measurement of NO3- in each batch of BRJ w/ or w/o NO3-: The NO3- content of each batch of 
BRJ w/ or w/o NO3- will be determined using high performance liquid chromatography (HPLC) 
(ENO-30, Eicom USA, San Diego, CA). 
Measurement of plasma NO3-, NO2-, and breath NO: Venous blood samples will be obtained prior 
to consumption of BRJ w/ or w/o NO3- and every hour thereafter for 3 hours, plasma rapidly 
separated by centrifugation, and frozen at -80° C until subsequently analyzed for NO3- and NO2- 
concentration using HPLC as described above. Breath NO levels will be measured at the same 
time points using a portable electrochemical analyzer (NIOX VERO, Circassia Pharmaceuticals, 
Chicago, IL). 
Measurement of skeletal muscle contractile function: A Biodex 4 system will be used to measure 
each subject’s muscle contractile properties as previously described.29-31 Briefly, subjects will 
perform maximal knee extensions with their dominant leg at angular velocities of 0, 1.57, 3.14, 
4.71, and 6.28 rad/s. (Not all older individuals may be able to achieve the highest velocity – if 
not, the highest measured angular velocity and associated torque will be used in all subsequent 
calculations.) The subject will perform 3-4 knee extensions at each velocity, with 2 min of rest 
allowed between each set of contractions. To eliminate artifacts, data will be “windowed” to 
isolate the isokinetic phase and smoothed using a 9 point weighted moving average filter. The 
highest torque generated at each velocity will be used to calculate peak power at that velocity, 
after which the resulting power-velocity curve will be fit to a polynomial function to determine 
the subject’s maximal knee extensor velocity (Vmax) and corresponding power (Pmax). 
Subsequently, the subject will perform an “all out” 50 contraction fatigue test (at 3.14 rad/s) to 
determine whether dietary NO3- influences fatigue resistance (i.e., increases average power) 
during repetitive, maximal activation. Finally, recovery of muscle function will be quantified by 
measuring restoration of torque during knee extensions performed periodically over the next 10 
min. 
 
Risks   
 
Likely: The insertion of an intravenous tube for blood drawing is associated with a small amount 
of discomfort. The subjects are likely to notice reddish urine or stools after drinking beetroot 
juice or control. This is normal and not harmful. 
 
Less Likely: The insertion of an intravenous tube for blood drawing is associated with a small 
risk of bleeding or infection. After the EKG, some people may experience a skin rash but this 
usually goes away without treatment. 
 
Rare: The risk of arrhythmia and heart attack are rare and the risk of death during exercise 
testing is extremely rare. The risk of heart attack is <0.1% in all patients undergoing exercise 
testing (and should be lower in these subjects since they have no coronary artery disease). The 
risk of death is reported as less than 2/10,000 tests or <0.02%.  
 
Mitigation of Risks   
 
Blood draw: Only trained staff will place IVs and collect blood samples 
 
EKG: trained nurses will prepare the skin for the EKG and place the electrodes. 

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Exercise test: Every effort to minimize these rare risks by observing and monitoring during 
testing. Emergency equipment and trained personnel are available to deal with any emergency. 
 
 
 
6.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
The following standard definitions will be used for this study: 
Adverse event (AE): Any untoward or unfavorable medical occurrence in a human study 
participant, including any abnormal sign (e.g. abnormal physical exam or laboratory finding), 
symptom, or disease, temporally associated with the participants’ involvement in the research, 
whether or not considered related to participation in the research. 
Serious Adverse Event (SAE): Any AE that results is place the participant at immediate risk of 
death from the event as it occurred, or results in inpatient hospitalization, prolongation of existing 
hospitalization, persistent or significant disability/incapacity, congenital abnormalities or birth 
defects, or death.  
Unanticipated problem: As Defined by DHHS 45 CFR part 46, any incident, experience, or outcome 
that meets all of the following criteria: 1) is unexpected, in terms of nature, severity, or frequency, 
given (a) the research procedures that are described in the protocol-related documents, such as 
the IRB-approved research protocol and informed consent document; and (b) the characteristics 
of the study population; 2) is related or possibly related to participation in the research (in this 
guidance document, possibly related means there is a reasonable possibility that the incident, 
experience, or outcome may have been caused by the procedures involved in the research); and 
3) suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
AEs will be graded according to the following scale: 
• 
Mild: An experience that is transient, and requires no special treatment or intervention. 
The experience does not generally interfere with usual daily activities. This includes 
transient laboratory test alterations. 
• 
Moderate: An experience that is alleviated with simple therapeutic treatments. The 
experience impacts usual daily activities. Includes laboratory test alterations indicating 
injury, but without long-term risk. 
• 
Severe: An experience that requires therapeutic intervention. The experience interrupts 
usual daily activities. If hospitalization (or prolongation of hospitalization) is required for 
treatment it becomes an SAE. 
Attribution of AEs and SAEs will be categorized as: 
• 
Not related: The AE is clearly not related to the study procedures (i.e., another cause of 
the event is most plausible and/or a clinically plausible temporal sequence is inconsistent 
with the onset of the event).  
• 
Possibly related: An event that follows a reasonable temporal sequence from the initiation 
of study procedures, but that could readily have been produced by a number of other 
factors. 
• 
Related: The AE is clearly related to the study procedures.  

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Reporting of AEs, SAEs, and unanticipated problems will follow the guidelines of the IU Standard 
Operating Procedure for Reporting Unanticipated Problems and Noncompliance. Specifically, the 
following events will be reported promptly (i.e., within five business days) to the IRB: 
• 
AEs (including SAEs) that are assessed by the PI or coinvestigators as (1) unexpected, (2) 
related or possibly related to participation, AND (3) suggests that the research places 
subject(s) or others at greater risk of harm than was previously known; 
• 
Major protocol deviations that may, in the opinion of the PI, (1) impact subject safety, (2) 
affect the integrity of the data, OR (3) affect study participant' willingness to participate 
in the study; 
• 
Noncompliance, which includes any action or activity associated with the conduct or 
oversight of the research that fails to comply with federal or state regulations, institutional 
policies governing human study participant research, or the requirements or 
determinations of the IRB. 
Unanticipated problems that do not meet the criteria for prompt reporting will be reported at time 
of protocol renewal to ensure the IRB has a full understanding of the conduct of the research  
Study Withdrawal/Discontinuation 
A participant may withdrawal from the study at any time verbally or by providing this request in 
writing as described in the informed consent document. As outlined in the consent document, if 
the participant/patient wishes to withdraw consent, the PI will: 
• 
no longer use and take reasonable steps to destroy all blood/tissue samples and 
information 
• 
not take back any research / analyses already completed  
7.0 
Statistical Considerations 
Statistical analyses: Normality of data distribution will first be tested using the D’Agostino-Pearson 
omnibus test. Analysis of individual endpoints will then be conducted using a mixed model analysis 
of variance (ANOVA) approach, with dose, trial number, and sequence as fixed effects and subject 
as a random effect to account for repeated measurements. As a preliminary analysis, carryover 
will be evaluated by testing the effect of sequence, i.e. does the effect of dose depend on trial. 
Assuming a sufficient washout period, i.e., no carryover effect, analysis of dose will proceed using 
all trials. Dose effects will be determined from ANOVA model results. A P value of <0.05 will be 
considered significant. 
Calculation of sample size/power analyses: Our proposed sample size (i.e., n=20) for this pilot, 
proof-of-concept study has been chosen primarily based on budgetary considerations and the 1 y 
duration of the project. G*Power© version 3.1.9.459 was used to determine the sample size 
required to provide a power (i.e., 1-ß) of 0.90 assuming an effect size (i.e., Cohen’s d) of 1.5. 
This effect size is based on the n=7 healthy older subjects that we have studied to date and is 
roughly the same as that we have previously observed for the effects of dietary NO3- on muscle 
contractile function in younger subjects.29,30 These calculations demonstrated that with α=0.05, 
n=15 would be sufficient to provide the chosen power. The power that a sample size of n=15 
would provide to detect other effect sizes is shown in Figure 4.  Based on these calculations, we 
will enroll 20 subjects to allow for subject drop-out. 
 

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Potential limitations and alternatives considered: A possible limitation of the proposed study is 
the lack of direct demonstration that dietary NO3- results in an increase in NO specifically within 
muscle. This is because 1) the biological half-life of NO is extremely short (i.e., seconds), 
precluding measurement of its concentration in human tissue samples, and 2) existing stable 
isotopic methods only measure the rate of NO production via the NOS pathway, and not the 
enterosalivary pathway. We will, however, measure changes in plasma NO3-/NO2- and breath NO 
(a biomarker of whole-body NO bioavailability) levels in response to dietary NO3-. We therefore 
do not consider the absence of direct measurements of plasma or muscle NO a significant 
limitation. 
A more significant limitation of the proposed 
study is that, due to budgetary and time 
constraints, we will be evaluating only two doses 
of NO3-. We will therefore be unable to identify 
the truly optimal dose (which could fall in-
between the two doses to be tested, or higher 
than the highest dose). As stated previously, 
however, there is very limited data in the 
literature 
regarding 
the 
dose-response 
relationship of dietary NO3-, especially with 
respect to changes in exercise performance. 
Thus, despite this limitation the information to 
be gained from this study will represent a significant advance, and in fact is a necessary first step 
towards the design of larger (e.g., multicenter) studies. 
By focusing specifically on changes in muscle contractile properties, we will not be able to 
determine whether dietary NO3- supplementation reduces the O2 cost of exercise in older subjects. 
We will also not be measuring the effect of acute and/or chronic dietary NO3- intake on muscle 
blood flow, or on muscle energetics or aerobic exercise performance. We will also not be testing 
whether dietary NO3- intake improves insulin sensitivity. Several recent studies, however, have 
reported that dietary NO3- (or NO2-) does not significantly improve aerobic exercise function in 
older men and women.60-63 (Despite increasing muscle contractile function.63) Similarly, several 
other recent studies have demonstrated that, contrary to the results of in vitro experiments,64 
dietary NO3- does not enhance insulin sensitivity in older humans.65-67 We therefore believe that 
we are well-justified in focusing on changes in muscle contractile properties, especially in light of 
the important role that reductions in muscle function play in the aging process. 
Because our focus is muscle contractile function, we considered using percutaneous electrical 
nerve stimulation rather than voluntary exercise. However, this approach inverts the normal 
orderly recruitment of motor units, preferentially depolarizing larger alpha motor neurons 
innervating type II muscle fibers.68 Thus, while potentially magnifying the effects of dietary NO3- 
on muscle speed and power it would significantly diminish the external validity of our findings. 
Although the proposed study will provide important new information regarding the effect of NO3- 
dose on muscle contractile function in older individuals, it will not address the mechanism(s) 
responsible for any improvements that may be observed. While clearly relevant, answering such 
questions are beyond the scope of the present investigation. 
Finally, we note that historically there have been concerns that increased NO3- intake may lead 
to formation of carcinogenic nitrosamines.69 However, the Joint FAO/WHO Expert Committee on 
Food Additives has concluded that “Overall, the epidemiological studies showed no consistently 

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increased risk for cancer with increasing consumption of NO3-.”63 Furthermore, diets high in 
vegetables, such as the DASH diet widely recommended to hypertensive patients, routinely 
exceed the WHO acceptable daily intake of NO3- by >5-fold. In contrast, the amount of dietary 
NO3- shown to improve exercise tolerance in previous studies is only slightly above the WHO 
limit.70 Nonetheless, the safety and efficacy of increased dietary NO3- would need to be established 
in follow-up large-scale, longer-term studies before BRJ or other NO3--rich foods or could be 
widely recommended to older individuals. 
 
8.0 
Privacy/Confidentiality Issues 
All study activities will be performed within areas respective of the participants’ right to privacy.  
Although there can be no absolute guarantee of confidentiality, every practical precaution will be 
taken. 
Each study participant will be assigned a unique ID. Samples and information will be de-identified 
using this unique ID. 
9.0 
Follow-up and Record Retention 
Study recruitment will be ongoing. The duration of the entire study is expected to be 12 months. 
Any remaining blood samples will be de-identified and discarded. The de-identified data will be 
retained on computer files indefinitely. Hard copy study documents will be kept in a locked, file 
cabinet in a locked room. Electronic study information will be stored on a specified, password-
protected network that is backed up daily. Only the study team and the relevant personnel will 
have access. Files will be kept on site until 2 years after study completion, and then sent to a 
secure archiving facility. Files will be kept for 7 years after study completion per state law 
requirements. 
 
10.0 References 
1. Projections of the Population by Selected Age Groups and Sex for the United States: 2010 to 
2050 (NP2008-T2) Population Division, U.S. Census Bureau (2008). 
2. Guralnik JM, Simonsick EM, Ferrucci L, Glynn RJ, Berkman LF, Blazer DG, Scherr PA, Wallace 
RB. A short physical performance battery assessing lower extremity function: association with 
self-reported disability and prediction of mortality and nursing home admission. J Gerontol 
1994;49:M85–M94. 
3. Kokkinos P, Myers J. Exercise and physical activity: clinical outcomes and applications. 
Circulation 2010;122:1637–1648. 
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