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The Open Nutraceuticals Journal, 2015, 8, 43-48 43
1876-3960/15 2015 Bentham Open
Influence of a D-aspartic Acid/Sodium Nitrate/Vitamin D3 Dietary
Supplement on Physiological Parameters in Middle-aged Men: A Pilot
Richard J. Bloomer*, Trint A. Gunnels, Ryan G. Moran and JohnHenry M. Schriefer
Cardiorespiratory/Metabolic Laboratory, Department of Health and Sport Sciences, University of Memphis, Memphis,
Abstract: D-aspartic acid (DAA), nitrate, and vitamin D3 have received considerable attention in recent years for their
potential health-enhancing properties. Using an open-label design, we evaluated the impact of a DAA/sodium nitrate/
vitamin D3 dietary supplement on blood testosterone and nitrate/nitrite, as well as subjective indicators of health, in
Methods: 10 overweight or obese men (mean age: 42 years) were assigned to ingest a DAA/sodium nitrate/ vitamin D3
supplement (either one or two servings per day) for 28 days. Blood total and free testosterone and nitrate/nitrite was
measured before and after 14 and 28 days of supplementation. Subjective assessment of to health indicators (e.g., energy
level, libido) was included at each collection time.
Results: Total and free testosterone increased on average 5-10%, which was not of statistical significance (p>0.05). The
response was highly variable; some men failed to respond to treatment, while men with relatively low basal testosterone
values experienced increases exceeding 20%. Plasma nitrate/nitrite was increased approximately 6-10 fold after treatment
with the supplement, with a trend noted for a time effect (p=0.07). Men reported a significantly better feeling following
supplement use, as evidenced by a time effect for both vitality (p=0.02) and libido (p=0.04), with a trend noted for
increased energy level (p=0.08) and mental outlook and mood (p=0.10).
Conclusion: Twenty-eight days of treatment with a DAA/sodium nitrate/ vitamin D3 dietary supplement increased blood
nitrate/nitrite and improved subjective feelings of vitality and libido in middle-aged men. In selected men with low basal
testosterone values, the supplement increased circulating levels of this hormone.
Keywords: D-aspartic acid, Dietary supplements, Libido, Sodium nitrate, Testosterone, Vitality.
Dietary supplements aimed at improving subjective
feelings of well-being in middle-aged and older men are of
great interest. This may be partly related to the fact that
decreased circulating testosterone is often associated with
aging , coupled with the fact that variables regulating
blood flow may be somewhat compromised by aging .
Related to the above, nutrients aimed at increasing
circulating testosterone and improving systemic blood flow
are of great interest. One ingredient that has received a great
deal of attention recently for its purported ability to increase
blood testosterone is D-aspartic acid (DAA). DAA is present
in rat nervous system and endocrine glands, and has been
shown to induce both growth hormone and luteinizing
hormone release , while stimulating the release of sex
hormones from the pituitary gland and testes in animals. For
*Address correspondence to the author at the Department of Health and
Sport Sciences, 106 Roane Fieldhouse, The University of Memphis,
Memphis, TN 38152, USA; Tel: 901-678-5638; Fax: 901-678-3591;
example, the incubation of DAA with isolated rat testes has
demonstrated an increased synthesis of testosterone ,
suggesting that DAA is involved in steroidogenesis.
Additional studies involving rodents have found similar
results [5, 6].
Much of the interest in DAA is based on the reported
42% increase in blood testosterone concentrations in young
(27-37 year old) men following daily supplementation at
3120 mg over the course of a 12 day period . Having said
that , both our lab (unpublished findings) and Willoughby
and Leutholtz  have noted conflicting findings to those of
Topo and colleagues; specifically, no increase in total or free
testosterone when DAA is used by young men (mean age
~24 years old) with relatively high basal testosterone. Aside
from this work, we are unaware of any human studies using
DAA, despite animal studies demonstrating increases in
testosterone with treatment [4, 6]. It is possible that DAA is
more helpful in middle-aged or older men, in particular those
with low basal testosterone values. This question is partly the
basis for the present pilot study.
44 The Open Nutraceuticals Journal, 2015, Volume 8 Bloomer et al.
Beyond DAA, recent work has indicated an association
between vitamin D3 and circulating testosterone [8, 9]. For
example, Pilz and colleagues reported that 31 healthy,
overweight men who were supplemented with approximately
3000 IU of vitamin D daily for one year had a significant
increase in total, bioactive, and free testosterone levels .
Based on these findings, coupled with those indicating
various health benefits of vitamin D3 supplementation ,
use of this nutrient may prove beneficial to men.
In addition to the use of DAA and vitamin D3 to elevate
blood testosterone, is the impact of sodium nitrate to increase
circulating levels of nitrate/nitrite in middle-aged men, and
the potential for improved blood flow and related outcomes
(e.g., enhanced energy levels). Indeed, dietary nitrate
ingestion has been reported to aid cardiorespiratory and
muscular endurance . Specifically, investigators have
used either sodium nitrate (~500mg) or beetroot juice
(containing nitrate at a dosage of approximately 500mg) as
an ergogenic aid, reporting improvements in exercise
performance , in addition to a lowering in blood pressure
. It has been suggested that these effects may be
mediated by an increase in nitric oxide (NO), which acts as a
vasodilator to open vessels and allow for greater blood flow.
In turn, greater oxygen and nutrient delivery to active tissues
(i.e., skeletal muscle) may be observed, leading to enhanced
feeling of energy and vitality.
Considering the above, the present pilot study sought to
determine the influence of a 28 day treatment period of
DAA/sodium nitrate/ vitamin D3 on subjective indicators of
health in middle-aged men, as well as blood testosterone and
nitrate/nitrite (the metabolites of nitric oxide).
MATERIALS AND METHODOLOGY
A total of 10 physically active men completed this study.
Subjects were not current smokers and were considered to be
in good overall health, without a history of cardiovascular or
metabolic disease (including hypertension). Subjects were
not using hormonal replacement therapy or dietary
supplements designed to increase hormone production; nor
were subjects using dietary supplements containing nitrate.
Subject characteristics are presented in Table 1. Health
history, medication and dietary supplement usage, and
physical activity questionnaires were completed by all
subjects and reviewed by an investigator to determine
eligibility. Subjects were informed of all procedures,
potential risks, and benefits associated with the study
through both verbal and written form. The study procedures
were approved by the University Institutional Review Board
(IRB) for Human Subjects Research. Subjects who
completed the study were compensated $150 for their time.
During the initial lab visit subjects completed all
paperwork, inclusive of the informed consent form.
Subjects’ heart rate and blood pressure, height, weight,
waist, and hip circumference were measured. Subjects were
provided with food logs and instructions regarding how to
complete these logs during the 48 hours before each test day.
Subjects returned to the lab on three occasions (before
supplementation, after 14 days of supplementation, and after
28 days of supplementation) to complete the assessments
indicated below. On each occasion subjects reported to the
lab following a minimum 10 hour overnight fast. Upon
arrival, subjects rested quietly for 20 minutes while seated in
a chair. Subjects then completed a questionnaire related to
their perceived level of energy and associated variables.
Their resting heart rate (via palpation of the radial artery)
and blood pressure (via auscultation) was measured. A blood
sample was taken and processed as described below. This
concluded their participation for the assigned day.
Following the above assessments on day 1, subjects were
evenly assigned to either one or two servings per day of the
supplement (D-Pol™; Purus Labs, Dallas, TX). The
supplement contained DAA at a dosage of 3120mg and
sodium nitrate at a dosage of 480mg. The supplement also
contained vitamin D3 at a dosage of 4000IU. The
Table 1. Characteristics of men assigned to one or two servings per day of the supplement.
One Serving (n=5)
Two Servings (n=5)
Body Weight (kg)
Body Mass Index (kg∙m-2)
Waist Circumference (cm)
Hip Circumference (cm)
Weekly Aerobic Training (hrs)
Weekly Anaerobic Training (hrs)
Values are mean±SEM.
No differences of statistical significance noted between groups (p>0.05).
D-aspartic Acid/Sodium Nitrate/ Vitamin D3 Supplement The Open Nutraceuticals Journal, 2015, Volume 8 45
supplement was provided in powder form and subjects were
required to mix the powder (approximately 5 grams) in water
(8-12 ounces) for consumption. Subjects assigned to one
serving per day were instructed to consume their daily
serving with breakfast. Subjects assigned to two servings per
day were instructed to consume their daily servings with
both breakfast and dinner. The supplement was provided to
each subject in a container and produced under standard
Good Manufacturing Practices by a dietary supplement
contract manufacturer. Subjects’ compliance to supplement
intake was determined at 100%, based on remaining powder
left in the supplement containers upon return.
Blood Collection and Analysis
Venous blood samples (~15mL) were taken from
subjects via needle and Vacutainer®. Samples were collected
before beginning supplementation (Pre) and after 14 and 28
days of supplementation. Following individual collection,
samples were processed accordingly and the plasma and
serum was stored at -70 ºC until analyzed. Samples for
analysis of total testosterone (catalogue: TE187S), free
testosterone (catalogue: FT178S), and estradiol (catalogue:
ES180S) were analyzed in serum using enzyme linked
immunosorbent assay (ELISA) procedures according to the
instructions provided by the manufacturer (Calbiotech, Inc.
Spring Valley, CA). Nitrate/nitrite was analyzed in plasma
using a commercially available colorimetric assay kit
(catalogue: 780001) according to the procedures provided by
the manufacturer (Caymen Chemical, Ann Arbor, MI). Prior
to each analysis, all samples were thawed and mixed
thoroughly, then analyzed in duplicate.
Due to the potential influence of circulating testosterone
and NO on health-specific parameters, a questionnaire was
designed by the investigators and completed by subjects on
the morning of each test day. Using a scale of 1-5, with 1
representing the lowest rating and 5 representing the highest
rating, subjects rated a number of variables pertaining to
overall health with regards to how they felt over the past two
Dietary Intake and Physical Activity
Subjects were instructed to consume their usual diet and to
record dietary intake during the 48 hours prior to each test
day. Detailed instructions for completing the records were
provided to each subject and records were analyzed using
nutritional software (Food Processor Pro; ESHA Research,
Salem, OR). Subjects were instructed not to consume alcohol
or caffeinated beverages or dietary supplements containing
caffeine or other stimulants, during the 48 hours prior to
each test day. In addition, subjects were asked not to engage
in sexual activity or to perform strenuous physical activity
for the 48 hours prior to each test day. Finally, subjects were
asked to obtain at least 7 hours of sleep the night prior to
teach test day.
Data were analyzed using a 2 (condition) by 3 (day)
analysis of variance (ANOVA). Tukey post-hoc testing was
used as needed. The data are presented as mean ± SEM. All
analyses were performed using JMP statistical software.
Statistical significance was set at p ≤ 0.05.
All 10 subjects successfully completed the four-week
study. One additional subject started the study but failed to
complete the final two weeks due to experiencing orthostatic
hypotension. We are uncertain whether or not this was due to
treatment with the supplement; however, since nitrate is
known as a vasodilator, it is possible that the supplement
contributed to this result. Data for this subject are not
Subject characteristics were not different between groups
(p>0.05), as can be viewed in Table 1. Subjects were in good
overall health, although all subjects were either overweight
or grade I obese based on BMI classification. Aside from a
higher vitamin C (p=0.03) and E (p=0.01) intake in the one
serving per day group, dietary intake was not different
between groups or across time (p>0.05; Table 2). Resting
heart rate was not different (p>0.05) but a time effect was
noted for systolic blood pressure (p=0.04; Table 3); groups
Table 2. Dietary data of men assigned to one or two servings per day of the supplement.
Vitamin C (mg)**
Vitamin E (mg)**
Vitamin A (RE)
Data are mean±SEM.
** Group effect for vitamin C (p=0.03) and vitamin E (p=0.01); One Serving > Two Servings.
No other differences of statistical significance noted (p>0.05).
Note: Values are for the 48-hour period immediately preceding each test day.
46 The Open Nutraceuticals Journal, 2015, Volume 8 Bloomer et al.
combined, day 14 lower than Pre; Two servings, day 14 and
day 28 lower than Pre.
With regards to subjects’ perceived feelings associated
with use of the supplement, a time effect was noted for
vitality (p=0.02) and libido (p=0.04), with day 14 and day 28
higher than Pre (Table 4). Trends for a time effect were
noted for energy level (p=0.08) and mental outlook and
No differences of statistical significance were noted for
blood testosterone or estradiol (p>0.05; Table 5). Of the five
men assigned to one serving per day of the supplement, three
experienced an increase in both total and free testosterone.
Of the five men assigned to two servings per day of the
supplement, two experienced an increase in both total and
free testosterone. A post-hoc power analysis indicated that
approximately 180 men would be needed to observe a
statistically significant effect for testosterone.
A trend for a time effect was noted for nitrate/nitrite
(p=0.07; Table 5), with values increasing 6-10 fold following
treatment with the supplement. With the exception of one
subject assigned to one serving per day of the supplement,
all men experienced an increase in nitrate/nitrite. A post-hoc
power analysis indicated that approximately 30 men would
be needed to observe a statistically significant effect for
Findings from the present study indicate that daily
ingestion of a DAA/sodium nitrate/ vitamin D3 supplement
for 28 days resulted in an increase in blood nitrate/nitrite and
improved subjective feelings of vitality and libido in middle-
aged men, with a trend for improving energy levels and
mental outlook and mood. In approximately 50% of men, the
supplement increased circulating levels of testosterone.
Table 3. Resting heart rate and blood pressure of men assigned to one or two servings per day of the supplement.
SBP (mm Hg)*
DBP (mm Hg)
Data are mean±SEM.
* Time effect for SBP (p=0.04); groups combined, day 14 lower than Pre (p=0.01); Two servings, day 14 (p=0.002) and day 28 (p=0.02) lower than Pre.
No other differences of statistical significance noted (p>0.05).
Note: HR=Heart Rate; SBP = Systolic Blood Pressure; DBP = Diastolic Blood Pressure
Table 4. Perceived feelings of men assigned to one or two servings per day of the supplement.
Mental Outlook & Mood†
Data are mean±SEM.
* Time effect for vitality (p=0.02); Day 14 (p=0.02) and Day 28 (p=0.01) higher than Pre.
* Time effect for libido (p=0.04); Day 14 (p=0.02) and Day 28 (p=0.04) higher than Pre.
No other differences of statistical significance noted (p>0.05).
† Trend for time effect for energy level (p=0.08).
† Trend for time effect for mental outlook & mood (p=0.10).
Note: Using a rating of 1-5, with 1 representing the lowest rating and 5 representing the highest rating, subjects were asked to rate each variable in the above
table, with consideration of how they felt during the prior two weeks.
D-aspartic Acid/Sodium Nitrate/ Vitamin D3 Supplement The Open Nutraceuticals Journal, 2015, Volume 8 47
These findings are in reference to a sample of middle-aged,
overweight or obese men.
Perhaps most importantly, our findings indicate that men
ingesting the DAA/sodium nitrate/ vitamin D3 supplement
reported improvements in multiple subjective measures of
physical and mental health, including vitality, libido, energy,
and mental outlook and mood (Table 4). These findings were
commonplace across our subjects, suggesting that the
supplement may be responsible for improving the general
outlook of middle-aged men with regards to these variables.
Of course, since this was an open label study, it is possible
that subjects were influenced by the placebo effect .
Additional, placebo controlled studies are needed using the
supplement in a sample of middle-aged men to replicate the
It is possible that the enhanced feelings noted were due in
part to the large increase in circulating NO metabolites
(nitrate/nitrite). The supplement resulted in an approximate
6-10 fold increase in nitrate/nitrite following treatment,
which is far larger than the increase observed with any other
dietary ingredient/supplement that we have reviewed. In fact,
in another pilot experiment in our lab using a sample of five
men (mean age: 29 years) who ingested one serving of the
supplement, plasma nitrate/nitrite was noted to be increased
approximately 10-fold above baseline when measured just
one hour following ingestion (unpublished findings). While
this increase is massive, we are uncertain of the physiologic
relevance of this change. It is possible that this increase is
representative of an increase in NO within the vasculature,
potentially influencing vascular homeostasis . This
would partly explain subjects’ ratings of improved energy
and associated variables, with treatment.
The increase in nitrate/nitrite, representative of NO, may
be responsible for the reduction in blood pressure noted with
supplementation-in particular within the two servings per
day group (Table 3). Considering that men in this study were
normotensive, it is certainly plausible to hypothesize that
men with hypertension may have responded to a greater
extent with treatment. Prior work involving nitrate ingestion
has noted blood pressure reduction , with the present
study confirming these findings in a group of normotensive
men. Based on the potential influence of nitrate ingestion on
blood pressure, care should be taken when dosing subjects
with nitrate, as too high of a dosage may result in too
significant a reduction in blood pressure. Additional work is
needed to determine the optimal dosage of nitrate for
purposes of blood pressure control.
Past work involving DAA supplementation has yielded
mixed findings with regards to increasing circulating
testosterone. Topo and colleagues noted an approximate 42%
increase in total testosterone following 12 days of DAA
supplementation in young (aged 27-37 years) men-a finding
that was observed in 20 out of 23 subjects . More
recently, Willoughby and Leutholtz reported that the same
dosage of DAA (~3 grams) taken for a 28 day period failed
to increase total or free testosterone in young (aged 18-23
years) men . We have recently noted similar findings as
Willoughby and Leutholtz, of no increase in total or free
testosterone when 24 young men ingested 3 grams daily of
DAA (unpublished findings). We believe that the age and
basal testosterone values of subjects may be responsible for
the discrepancy in findings. For example, our prior work
sample consisted of a group of young men between the ages
of 18-39 years whose total testosterone levels were in the
mid or high range of “normal” (mean: 6.2 ng∙mL-1);
considering the normal range of ~ 3 - 9 ng∙mL-1 .
Subjects in the study of Willoughby and Leutholtz  were
also young (18-23 years), with a mean basal total
testosterone value of 8.1 ng∙mL-1. This is compared to
subjects in the study of Topo and colleagues , who ranged
in age from 27 to 37 years and had a mean basal testosterone
value of 4.5 ng∙mL-1. The lower mean basal testosterone
value of subjects in the Topo et al. study could have been
responsible for the differing findings for testosterone
elevation with DAA treatment. Considering the low mean
basal testosterone values of subjects in the present study
(e.g., ~3 ng∙mL-1), it is not surprising that we observed a
small increase in this measure, with some subjects who were
very low to begin with experiencing a more pronounced
effect with DAA treatment. More work is needed to
determine if DAA supplementation is beneficial for men
with depressed basal testosterone concentrations.
Aside from the focus on testosterone, reports from animal
models indicate that DAA may lead to an increase in
estradiol-a result of increased aromatase activity and the
conversion of testosterone to estradiol [18-20]. A lack of
increase in estradiol has been noted following DAA
supplementation in the work of Willoughby and Leutholtz
, as well as in our recent work (unpublished findings). The
present data extend those findings, noting minimal change in
Table 5. Biochemical data of men assigned to one or two servings per day of the supplement.
Testosterone Total (ng∙mL-1)
Testosterone Free (pg∙mL-1)
Data are mean±SEM.
No differences of statistical significance noted (p>0.05).
† Trend for time effect for Nitrate/Nitrite (p=0.07).
48 The Open Nutraceuticals Journal, 2015, Volume 8 Bloomer et al.
estradiol, even with 6 grams daily of DAA over a four week
In summary, twenty-eight days of treatment with a
DAA/sodium nitrate/ vitamin D3 dietary supplement
increases blood nitrate/nitrite and can improve subjective
feelings of vitality and libido in middle-aged men. In men
with low basal testosterone values, the supplement may
increase circulating levels of this hormone. Additional work
is needed, using a placebo controlled design and a larger
sample of men, to more fully elucidate the role of
DAA/nitrate/ vitamin D3 to improve selected measures of
health in middle-aged to older men.
CONFLICT OF INTEREST
Funding for this work was provided in part by Formulife,
Inc. RJ Bloomer has been a principal investigator on
research projects and a consultant to dietary ingredient and
supplement companies. No other author declares any conflict
Funding for this work was provided in part by Formulife,
Inc. and the University of Memphis. The authors
acknowledge the assistance of Ramzi Majaj in performing
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Received: November 19, 2014 Revised: February 11, 2015 Accepted: February 12, 2015
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