Dietary Nitrate and Muscle Power With Aging
Phase: NA
Conditions: Aging, Sarcopenia
Sponsor: Indiana University

 
Protocol Version 04Jan2022 
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Dietary Nitrate and Muscle Power with Aging 
 
 
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. 
 
 
 
 
Tarah Ballinger, M.D. 
Ziyue Liu, Ph.D. 
 
 
 
 
Edgar Gallardo 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Protocol Version 04Jan2022 
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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 
 
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 (1,2). Thus, any intervention that significantly enhances muscle 
contractile properties 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, with age-
related changes in the size, properties, and neural control of muscle likely all playing a 
role (3). Another important factor, however, may be a fall in nitric oxide (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 (cf. Refs. 4-8 for review). Specifically, 
during isometric contractions NO may, or may not, slightly suppress maximal force 
production (4-8). This may be due to nitrosation or S-nitrosylation of various proteins 
(9). During concentric activity, however, NO significantly increases the rate of force 
development, maximal shortening velocity, and maximal power of both single muscle 
fibers and isolated muscles (4-8).  
 
Based on animal studies, these stimulatory effects are thought to be mediated via 
activation of the classic NO-sGC-cGMP pathway (10), and have been euphemistically 
described by Maréchal and Gailly (5) as a “slow-to-fast” shift qualitatively akin to the 
chronic transformation of muscle fiber type that occurs with, e.g., prolonged electrical 
stimulation. 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- (11,12). In skeletal muscle itself, there is a dramatic 
decrease in interstitial NO2- and NO3- concentrations (13), as well as a reduction in 
intracellular NO3- content (14) These changes are accompanied by a decline in the 
activity of the neuronal form of NO synthase (nNOS, or NOS1) (15,16), the primary 
isoenzyme within muscle responsible for the synthesis of NO from L-arginine, O2, and 
NADPH. This is associated with an age-related reduction in flow-mediated vasodilation 
(17-20), 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 (21). Taken together, 
these data suggest that decreased skeletal 
muscle 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 
Figure 1. NO-sGC-cGMP signaling via the enterosalivary. From 
Ref. 22. 

 
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is now recognized that NO3- in the diet is a significant source of NO in body (22-26) 
(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 up to ~25% of basal whole-body NO production 
(27,28). 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 (22-26), 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 NO3-/NO2-/NO metabolism (29). 
 
Haider and Folland (30) and Whitfield et al. (31) have reported that dietary NO3- intake 
enhances the rate of force development and peak force output of the human quadriceps 
muscle during electrically-stimulated contractions. Numerous recent studies (reviewed in 
32) demonstrate that dietary NO3- can enhance other types of exercise performance in 
various subject groups. To date, however, only a handful of studies have specifically 
examined the impact of dietary NO3- on exercise capacity in older individuals (33-35). 
These studies found no effect of consuming 9.6-12 mmol NO3-/d for 3-7 days on various 
measures of aerobic exercise performance in healthy older men and women (33,34) or 
in older individuals with hypertension or heart failure (HF) (35). Similarly, Justice et al. 
(36) reported that ingesting NO2- (not NO3-) at 1.2-2.4 mmol/d for 10 weeks did not 
improve VO2max in middle-aged and older study participant, although it did increase the 
rate of force development (but not maximal force, i.e., strength) during isometric muscle 
contractions. Importantly, however, none of these studies of older study participant 
measured maximal muscle power. The effects of dietary NO3- on this critical aspect of 
muscle contractile function in older study participant are therefore still completely 
unknown. Moreover, it cannot be automatically assumed that dietary NO3- will be equally 
efficacious in improving muscle function in older persons as we have found in other 
study participant groups (37-39). For example, in rodents, the effects of dietary NO3- are 
more prominent in fast- vs. slow-twitch muscle (40,41), and some (42-45), but not all 
(46-48), 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 
muscle (49) could limit the beneficial effects of dietary NO3- on muscle NO-sGC-cGMP 
signaling in older persons 
 
Even if it can be shown that dietary NO3- acutely improves muscle function in older men 
and women, it remains to be established whether such benefits can be sustained, 
and/or whether such effects have a positive impact on a person’s daily life. This is not 
necessarily a given, as it is well known that tolerance develops quite quickly in response 
to pharmacological nitrates, e.g., glyceryl trinitate (nitroglycerin) (49). This does not 
seem to be true, though, for dietary NO3- (50,51) – in fact, animal studies indicate that 
ingestion of NO3- for 5-7 days may lead to positive adaptations in calcium handling 
proteins and hence force production in muscle that would tend to mitigate age-related 
declines in these parameters (40). Notably, however, a recent study of humans was 
unable to reproduce these findings (31). 

 
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Our Preliminary Data suggest that the effects of dietary NO3- on muscle speed and 
power are maintained (but not enhanced) for at least 14 days with repeated (i.e., daily) 
dosing in humans (see below). 
 
Preliminary data  
Using methods essentially identical to those to be employed in the proposed 
experiments, including use of a “gold standard”, double-blind, placebo-controlled, 
crossover design, we previously demonstrated that 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 (37). 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 (38). Lastly, we found 
even greater increases in Vmax and Pmax (of 12±5%, P=0.09, and 13±4%, P<0.05, 
respectively) in nine middle-aged patients with systolic heart failure (HF) (52). The 
greater beneficial effect of dietary NO3- in HF patients presumably reflects the fact that, 
similar to healthy aging, HF results in reduced NO bioavailability (53). 
 
Using the same methods, we have also recently determined the effects of acute dietary 
NO3- intake in seven men and women 61-78 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 146±19%, 
respectively, by 2 hours after ingestion, and all three remained elevated for the 
remainder of the experiment. These observations are consistent with previous research 
in older study participant demonstrating that plasma NO3- and NO2- concentrations peak 
2-3 hours after ingestion of a NO3- load (54), which is comparable to the time course 
observed in younger study participant (55-57).  
  
 
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 
exer. 
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).   

 
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As shown in Fig. 2, dietary NO3- intake increased Vmax in 
six out of seven of these study participants, with the 
average improvement being 9±3% (P<0.05). Pmax 
increased in five out of the seven study participants, 
with the average increase being 4±3% (P=0.15). 
These preliminary results, which were presented at 
the 64th annual American College of Sports Medicine 
meeting in Denver, CO, May 30-June 3, 2017, are 
similar to those we obtained previously in younger 
individuals (37), and thus support our primary 
hypothesis. 
 
Biopsy samples were obtained from the v. lateralis 
of two of these study participant ~3 h after 
ingestion of either the placebo or NO3- and analyzed 
for cGMP content.  As shown in Fig. 3, muscle cGMP 
was higher in both study participants in the NO3- 
trial. These data support our hypothesis that dietary NO3- intake 
enhances muscle contractile function by increasing muscle NO-sGC-
cGMP signaling.    
 
Another of the study participants (number 5) 
continued to ingest either the NO3- supplement 
or the placebo daily for a total of 14 days, 
separated by a 10 day washout period. As 
shown in Fig. 4, their Pmax and Vmax were 
enhanced almost identically after both acute and chronic ingestion, 
such that the two dashed curves are superimposable. These data 
demonstrate that tolerance does not develop in response to 
repeated ingestion of dietary NO3-. Furthermore, since Vmax and 
Pmax were comparable after 14 vs. 1 day of placebo treatment (i.e., 
25 vs. 11 day post NO3- treatment), they also indicate that even a 10 
day washout period (vs. the 14 days we propose to use) is sufficient 
to eliminate any carryover effects. 
 
In summary, we have demonstrated that dietary NO3- improves 
maximal muscle speed and power in a variety of subject populations, 
including a small number of healthy, elderly men and women. 
Furthermore, we have demonstrated that dietary NO3- intake results 
in an increase in muscle NO-sGC-cGMP signaling. Finally, we have 
demonstrated that daily ingestion of dietary NO3- for 14 days does 
not result in tolerance. These preliminary data therefore provide very 
strong support for the current study. 
 
2.0 
Rationale and Specific Aims 
 
We hypothesize that increased dietary NO3- intake will enhance muscle NO-sGC-cGMP 
signaling in older men and women, leading to an increase in maximal speed and power. 
Figure 3. Effect of 
NO3- ingestion on 
muscle cGMP 
content. 
Fig. 2 Changes in maximal knee 
extensor speed (Vmax, top panel) 
and power (Pmax; bottom panel_ 
in response to dietary NO3-; intake 
in older adults  
 
Figure 4. Effects of acute vs. 
chronic dietary NO3- intake on 
maximal knee extensor power 
(Pmax) and speed (Vmax) in a 
78 y old woman. 
0%
20%
40%
60%
80%
100%
120%
140%
160%
Subject 1
Subject 3
[cGMP] (% of placebo)

 
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Helping drive our hypotheses are the consistent improvements in muscle speed and 
power that both we and others have observed following dietary NO3- intake in healthy 
younger study participants (37), athletes (38,39), and especially heart failure patients 
(52).  
 
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 study participant. 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. In preliminary experiments, however, we have not been able to reproduce 
the O2-sparing effect of NO3- ingestion, and several recent studies have reported that 
dietary NO3- (or NO2-) does not significantly improve aerobic exercise function in older 
men and women (32-35). Similarly, several other recent studies have demonstrated 
that, contrary to the results of in vitro or animal experiments, dietary NO3- does not 
enhance insulin sensitivity in older humans (58-60). We therefore believe that we are 
well-justified in focusing on changes in muscle contractile properties, especially in light 
of our preliminary data and 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 (cf. 61 for review). Thus, while 
potentially magnifying the effects of dietary NO3- on muscle speed and power it would 
significantly diminish the external validity of our findings. 
 
We considered, but rejected, various alternatives to our “gold standard”, double-blind, 
placebo-controlled, crossover design. This design is the most efficient for testing all of 
our hypotheses, i.e., it requires the fewest number of study participant to provide 
adequate statistical power (since each subject serves as their own control). A cross-
sectional study would require >10x study participant to provide equivalent power for 
detecting changes in Pmax.  
 
Finally, we note that historically there have been concerns that increased NO3- intake 
may lead to formation of carcinogenic nitrosamines (62). However, the Joint FAO/WHO 
Expert Committee on Food Additives has concluded that “Overall, the epidemiological 
studies showed no consistently 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 (63). 
Nonetheless, the safety and efficacy of increased dietary NO3- would need to be 
established in follow-up large-scale, longer-term studies before beet root juice (BRJ) or 
other NO3--rich foods could be widely recommended to older individuals. 
 

 
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Specific Aim #1: 
Determine the acute effects of dietary NO3- intake on 
muscle NO-sGC-cGMP signaling and muscle contractile 
properties 
 
Specific Aim #2: 
Determine the cumulative or chronic effects of dietary 
NO3- intake on muscle NO-sGC-cGMP signaling and muscle 
contractile properties 
 
Exploratory Aim #3: 
Assess the effects of dietary NO3- supplementation on 
perceived fatigue, physical function, and physical activity 
during daily life via questionnaires and accelerometer data  
 
 

 
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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, EKG, and routine blood tests 
• 
Agrees to also participate in the FIT study (IRB # 1707550885).  
 
Exclusion: 
• 
Men and women <65 or >79 years of age 
• 
Unable to provide informed consent 
• 
Currently pregnant or lactating 
• 
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), neuromuscular disease (e.g., cervical spondylotic radiculomyelopathy, 
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 
• 
Expending greater than 1500 kcal/week in moderate or greater intensity physical 
activity as defined by the CHAMPS physical activity questionnaire 
 
One hundred study participants will be enrolled to attain sixteen completed participants.  
 
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), 
NIH sponsored website/email via ResearchMatch.org and newspaper advertisements.   
In addition, our study will be listed on the Indiana CTSI study listing called the All IN for 
Health TrialX iConnect. iConnect is a HIPAA-compliant and secure public facing research 
recruitment platform provided by the Indiana Clinical and Translational Sciences Institute 
(Indiana University) consisting of a study trial listing and volunteer registry. This system 
is licensed by Indiana University from TrialX and is monitored by the Indiana University 
Information Technology Services (UITS) and University Information Security Office (ISO). 
Study Teams have access to their study listings and participant referrals based on a secure 
login. There is a small Administrative Team within the Indiana CTSI that manages the 
iConnect platform and has access to all study information, referrals, and registry 
volunteers. This online platform provides researchers with the ability to create a public 
facing webpage that can contain a brief prescreener and the ability for the public to refer 
themselves to the trial: (https://research.indianactsi.org). The study listing information 
can be modified based on the study and targeted population and provides a direct, 

 
Protocol Version 04Jan2022 
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easily managed system to enhance communication with the public. Study teams can also 
use this webpage as the landing or referral page for any marketing materials that are 
being used (either by listing the hyperlink or creating a scannable QR code). We have 
included a screenshot of our study listing for your review and approval. Please see 
attachment titled "Screenshot iConnect". In addition, potential participants can complete 
a pre-screener that will determine their preliminary eligiblilty (see attachment “Pre-
screener for iConnect.”) 
The study listings are open to the public and any persons who visit our study page may 
submit their contact information to us through the listing. We can access that information 
by logging in to our iConnect dashboard. We may respond to and contact interested 
volunteers directly through the platform or by university phone or email. We will use the 
following guide for any email responses for phone conversations with the volunteers. For 
our standard messaging, please see attached "iConnect Study Team Reply Email 
Message." 
In addition to the study listing(s), potential participants will be identified from the Indiana 
CTSI All IN for Health voluntary registry IU IRB Protocol Number 1105005444. This 
registry was created so that interested individuals could create a volunteer profile to 
receive information about ongoing studies. Persons in this registry have provided their 
self-reported health information for the purposes of being matched to appropriate 
research studies. The registry volunteers have signed an electronic consent/authorization 
so that their information can be used and they can be contacted for this reason. 
We can also match participants in the registry and are able to send out a message to them 
through the system in a de-identified manner. We may use the search and filter tool in 
the system to identify a group of volunteers who match our study criteria based upon the 
information that the volunteers have included in their profiles. We may then send an 
outreach message to those de-identified matched volunteers through the online system. 
If we send an outreach message to matched volunteers, we will use standard messaging 
(please see attached "iConnect Volunteer Outreach Email Message Template." Matched 
volunteers who receive our outreach message and indicate they are interested and agree 
to be contacted specifically for this study will approve for their contact information to be 
accessible by us through the iConnect portal. If we contact those interested matched 
volunteers by email or phone, we will use the language from Template C [file name of 
“iConnect Study Team Reply Email Message” for any email responses for phone 
conversations with the matched volunteers. 
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 
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. Subjects will be asked 

 
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to fast for 12 hours prior to their screening visit, before they sign 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 by the study statistician. 
A separate consent form for participation in the Fit Core (IRB study #1707550885) will be 
administered by the Fit Core staff according to their policies and procedures. 
 
 
5.0 
Study Procedures 
 
Study Design 
 
A diagram of the flow of the study for each participant is shown in Figure 6. 
 
 
Each participant will be studied using a double-blind, placebo-controlled, crossover 
design.  
The dose of NO3- to be given, which is roughly the equivalent of 3-4 whole beets or 2 
cups of cooked spinach (64), has been chosen based on our previous results (37,38,52) 
and preliminary data, as well as a recent study by Wylie et al. (55) demonstrating that, 
acutely (i.e., as a single dose), at least 8.4 mmol are needed to elicit an improvement in 
exercise performance but additional improvement does not result from a dose of 16.8 
mmol. The placebo, also to be purchased from James White Drinks Ltd., is prepared by 
extracting NO3- from beetroot juice (BRJ) using an ion exchange resin and is 
indistinguishable in packaging, color, taste, texture, and smell from the standard 
product. Importantly, this placebo does not alter plasma NO3-/NO2- or breath NO 
concentrations or the physiological responses to exercise. 
 
Study Procedures 
 
On Visit 1 (Pre-test), all study participants will complete a screening examination: 
questionnaires will be completed regarding their physical activity and perceived fatigue 
and physical function.  The participant will undergo a complete history and physical 
exam by a study physician, an EKG, and phlebotomy for screening and laboratories 
(complete blood count, liver and kidney function tests, electrolytes, fasting glucose and 
insulin), and an urine test to assess kidney function. They will also practice the entire 

 
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isokinetic dynamometer exercise test. Participants will then be escorted to the Fit Core 
where Fit Core staff will consent and then complete the Fit Core protocol (IRB study 
#1707550885). 
 
Participants will be instructed to consume their normal diet (aside from the BRJ 
supplement) throughout the study. They 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 hours; 
Ref. 27) 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 U.S. 
(54) has no significant influence on plasma NO3- levels (54). Participants will be asked to 
refrain from using chewing gum, alcohol, and caffeine-containing food/drinks for 24 
hours 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 (65,66). Participants will be 
asked to fast for 12 hours prior to each remaining study visit. 
On Visit 2 (Day 1 pre-crossover), study participants will complete questionnaires 
(PROMIS® questionnaire) regarding their perceived fatigue and physical function, will 
have their vital signs assessed, and will undergo phlebotomy for NO3- and NO2- 
measurement.   They will then ingest 140 mL of BRJ either containing or depleted of 
11.2 mmol of NO3- (i.e., BRJ w/ NO3- or BRJ w/o NO3-). After allowing 2 hours for 
absorption, they will undergo the isokinetic dynamometer. After completion of the 
dynamometer assessment, subjects will complete the Fit Core protocol, less the DEXA 
scan.  Blood samples will be obtained at hourly intervals during the study. Heart rate 
and blood pressure will be measured at the same scheduled times as blood samples 
after which a final blood sample will be obtained. The sequencing of the measurements 
have been chosen to minimize potential interference/carryover effects that might 
influence the data and/or its interpretation.   
Following completion of the above tests, the participant will continue to ingest 140 mL 
of BRJ w/ NO3- or BRJ w/o NO3- every morning for an additional 13 days, after which the 
protocol described above will be repeated on the 14th day.  To encourage compliance 
with the experimental protocol and enhance subject retention, the subjects will be 
initially provided with only a 7 day supply of BRJ. They will also be asked to keep and 
return the empty BRJ bottles at the next visit. 
On Visit 3 (Day 7 pre-crossover), participants will be asked to return the empty BRJ 
bottles. At this visit, participants will have their vital signs assessed and a blood sample 
will be obtained to quantify plasma NO3- and NO2- concentrations.  Participants will be 
asked to rate their perception of fatigue and physical function during the previous week 
using the NIH-funded Patient-Reported Outcomes Measurement Information System 
(PROMIS®). The final 7 days of BRJ will be provided. They will also be asked to keep 
and return the empty BRJ bottles at the next visit. 
On Visit 4 (Day 14 pre-crossover), participants will be asked to return to undergo the 
same studies as described above for Study Day 2. In addition, participants will be asked 
to rate their perception of fatigue and physical function during the previous week using 
the NIH-funded Patient-Reported Outcomes Measurement Information System 

 
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(PROMIS®).  The study team member will also complete BRJ accountability for the 2nd, 
7-day consumption period by collecting the empty BRJ bottles 
Then the study participant will undergo a 14 day (minimum) washout period consuming 
neither either BRJ w/ NO3- nor BRJ w/o NO3-. After this, the study participant will be 
crossed-over to receive whichever treatment they did not receive first. 
On Visit 5 (Day 1 post-crossover), the study participant will undergo the same 
procedures as described for Study Day 2. 
On Visit 6 (Day 7 post-crossover), the study participant will undergo the same 
procedures as described for Study Day 3 
On Visit 7 (Day 14 post-crossover), the study participant will undergo the same 
procedures as described for Study Day 4. 
During each 14 day intervention period (i.e., between Study Days 2 and 4 and 5 and 7 
inclusive), an Actigraph wGT3X-BT activity monitor will be worn at the hip during all 
waking hours except when bathing or swimming. The Actigraph measures ambient light 
and also accelerations in all three directions, from which total activity counts, active 
time, sedentary time, sleep, etc., are estimated/quantified. 
 
Subjects will complete a log indicating the times that they wore the activity monitor and 
return it to the study staff at each visit. 
 
Study Methods 
 
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-: 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.  
 
 
Measurement of skeletal muscle contractile function: A Biodex 4 system will be used to 
measure each subject’s muscle contractile properties as previously described (30,33). 
Briefly, study participant 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 achieved 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 minutes 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 using the 
manufacturer’s software. 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 with a parabolic function to determine the subject’s Pmax and Vmax (i.e., Y-

 
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maximum and 2nd Y-intercept of fitted parabola, respectively) as previously described 
(30,33). 
 
Measurement of perceived fatigue, physical function, and physical activity: Perceived 
fatigue and physical function will be assessed using PROMIS® Short Forms 8a and 20a, 
respectively , which are physical functioning questionnaires. Actual physical function will 
be quantified by the FIT Core using a suite of tests, i.e., usual gait speed, fast gait 
speed, Short Physical Performance Battery, 30 s chair stand performance, 6 min walk 
distance. Physical activity will be quantified using ActiGraph wGT3X-BT activity monitors 
(ActiGraph, Pensacola, FL). On Study Day 1, each participant will be given a fully-
charged monitor and directed to wear the monitor on their right hip (superior to the iliac 
crest) for the next 7 days, removing it only when sleeping or during aquatic activities 
(i.e., bathing, swimming), and to complete a log of when the monitor is worn. On day 7, 
they will exchange the monitor for another, to be worn for the next 7 days. Upon return 
of the monitors, the data will be downloaded using the ActiLife 6 software, divided into 
60 second epochs, and analyzed to quantify both total and bouted (i.e., >10 min 
duration) sedentary behavior, light physical activity, and moderate-to-vigorous physical 
activity (<100 counts/min, 100-2020, and >2020 counts/min, respectively). Total counts 
will also be recorded, since this may be more closely associated with the health benefits 
of physical activity (68). Results will be considered valid only if each monitor is worn for 
at least 10 hours/day over at least 4 days (including at least one weekend day) each 
week. 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  
 
Beetroot Juice: Beetroot juice consumption or the consumption of the BRJ placebo may 
cause stool and/or urine to appear pink. This does NOT indicate bleeding. You may 
experience mild gastrointestinal distress (cramps, bloating) or diarrhea following 
ingestion of the BRJ. In rare cases there is a theoretical increased risk of upper GI 
cancers if a compound called “nitrosamine” is made from nitrates by the body.  
However, in large studies of many people followed up for many years, a fruit and 
vegetable-rich diet (where the acceptable daily intake of nitrates was exceeded several-
fold) was not associated with any increase in cancer or mortality.  The 2003 Joint Food 
and Agricultural Organization/World Health Organization Expert committee concluded 
that there was no evidence that nitrates are carcinogenic to humans. 
 
Questionnaires: You may experience emotional discomfort when answering some 
questions in the health questionnaires.  If any particular question makes you 
uncomfortable, you may discuss its importance and the need to answer it with the 
specially trained interviewer.  You may choose not to answer any question with which 
you still feel uncomfortable. 
 

 
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Needle Stick: Slight pain, bruising or bleeding can occur. Rarely, infection can occur. 
Very infrequently, faintness may occur. To minimize the discomfort and risks associated 
with blood draw, only trained staff will collect blood samples. 
 
Exercise Test: Your muscles may feel fatigued during the exercise test. You may also 
develop soreness in your muscles or joints. Very rarely, an exercise test, such as the 
neuromuscular function test, may be associated with serious complications including, 
but not limited to: fainting and disorders of the heart beat (too fast or too slow) which 
may require hospitalization; heart attack, stroke, or death; and muscle or joint pain.  We 
will make every effort to minimize these rare risks by observing and monitoring during 
testing.  However, no guarantees can be made.  Emergency equipment and trained 
personnel are available to deal with any emergency. 
 
EKG: some people may experience a skin rash but this usually goes away without 
treatment. 
 
Fit Core: the risks for the Fit Core are described in that protocol (IRB study 
#1707550885). 
 
Another risk of this study is the possible loss of confidentiality, which is minimal. Even 
though the risk is small, a link exists between your protected health information and 
your sample. In addition to the risks listed above, there may be some unknown or 
infrequent and unforeseeable risks associated with participation in this study. You will be 
informed in a timely manner of any new information, findings or changes to the way the 
research will be performed that might influence your willingness to continue your 
participation in this study. 
 
Mitigation of Risks   
 
Questionnaires: Subjects may feel uncomfortable answering portions of the 
questionnaires. If so, they may discuss the importance of questions and the need to 
answer them with a member of the research team.  They may choose not to answer any 
question with which they still feel uncomfortable. 
 
Blood draw: Only trained staff will place IVs and collect blood samples 
 
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. 
 
EKG: trained nurses will prepare the skin for the EKG and place the electrodes. 
 
Fit Core: the mitigation of risks for the Fit Core are described in that protocol (IRB study 
#1707550885). 
 
These instructions will be provided both verbally and in written form.  
 

 
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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: 
 

 
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• 
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.  
 
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. 
 
7.0 
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  
 
8.0 
Statistical Considerations 
 
Data analyses: The primary outcomes are muscle contractile function (i.e., Vmax and 
Pmax) and muscle cGMP concentration on day 1 and day 14, as separate outcomes. H0 
is that they are not different while on BRJ w/ NO3- versus on BRJ w/o NO3- and Ha is that 
they are greater while on BRJ w/ NO3-. This hypothesis will be tested using a mixed 
model analysis of variance approach, with treatment (NO3-, placebo), sequence (NO3--to-
placebo, placebo-to-NO3-), and intervention period (1st, 2nd) as fixed effects and subject 
as a random effect to account for repeated measurements. Carryover will be evaluated 
by first testing the effect of sequence, i.e., does the effect of treatment depend on 

 
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sequence. Assuming no carryover, analysis of treatment will proceed using both sets of 
data. The perceived fatigue, physical function (both obtained from the Fit Core 
protocol), and accelerometer data will be analyzed using functional and time series data 
analysis techniques. A two-sided P<0.05 will be considered significant. SAS 9.4 will be 
used for data analysis (SAS Institute, Cary, NC). 
 
Sample size calculations: Samples size calculations were performed based on our 
preliminary data on NO3--induced changes in Pmax in seven older study participant. 
Based on the observed effect size of 0.47 and assuming an α of 0.05, N=10 would be 
required to achieve a power (1-β) of 0.80 and N=16 would be required to achieve a 
power of 0.90. To allow for screen failures and potential withdrawals, we will therefore 
enroll 100 study participants (to finish 16). 
 
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 the muscle (or 
plasma) samples, and 2) existing stable isotopic methods only measure the rate of NO 
production via the NOS pathway. We will, however, measure changes in  plasma NO3-
/NO2- levels in response to dietary NO3-. We therefore do not consider the absence of 
direct measurements of muscle NO to be a significant limitation. 
 
A potentially more important limitation to this research is the lack of pilot data for 
certain measurements. Thus, although we will have adequate power to detect increases 
in Vmax and Pmax), we cannot be assured that we will be able to detect differences in 
all of our endpoints. However, the proposed experiments would still provide the data 
needed to design a larger study that is adequately powered to detect changes in all 
variables.  
 
9.0 
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. 
 
10.0 Follow-up and Record Retention 
 
Study recruitment will be ongoing.  The duration of the entire study is expected to be 24 
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 

 
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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. 
 
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