The Journal of Nutrition

Nutrition and Disease

Inorganic Nitrate and Beetroot Juice

Supplementation Reduces Blood Pressure
in Adults: A Systematic Review and
Meta-Analysis1–3
Mario Siervo,* Jose Lara, Ikponmwonsa Ogbonmwan, and John C. Mathers

Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019

Human Nutrition Research Centre, Institute for Ageing and Health, Newcastle University, Campus for Ageing and Vitality,
Newcastle on Tyne, UK

Abstract
Diets including food products rich in inorganic nitrate are associated with lower blood pressure (BP). The evidence for the
BP-lowering effects of inorganic nitrate and beetroot in randomized clinical trials has not been systematically assessed.
The objective was to conduct a systematic review and meta-analysis of randomized clinical trials that examined the effects
of inorganic nitrate and beetroot supplementation on BP. Medline, EMBASE, and Scopus databases were searched from
inception to February 2013. The specific inclusion criteria were: 1) randomized clinical trials; 2) trials reporting effects on
systolic or diastolic BP or both; and 3) trials comparing inorganic nitrate or beetroot juice supplementation with placebo
control groups. Random-effects models were used to assess the pooled BP effect sizes. Sixteen trials met the eligibility
criteria for the systematic review. All studies had a crossover study design. The trials were conducted between 2006 and
2012 and included a total of 254 participants with 7–30 participants/study. The duration of each intervention ranged from
2 h to 15 d. Inorganic nitrate and beetroot juice consumption were associated with greater changes in systolic BP [24.4 mm Hg
(95% CI: 25.9, 22.8); P < 0.001] than diastolic BP [21.1 mm Hg (95% CI: 22.2, 0.1); P = 0.06]. The meta-regression
showed an association between daily dose of inorganic nitrate and changes in systolic BP (P < 0.05). Inorganic nitrate
and beetroot juice supplementation was associated with a significant reduction in systolic BP. These findings need to
be tested in long-term trials and in individuals at greater cardiovascular risk. J. Nutr. 143: 818–826, 2013.

Introduction
Hypertension exceeds smoking as a causal factor in total attrib-
utable mortality (1). Globally, two-thirds of stroke and one-half
of ischemic heart disease events are linked to non-optimal blood
pressure (BP)4 control, which contributes to ~13% of all deaths
and 4.5% of all disability-adjusted life years (1).
Antihypertensive drugs have attenuated the adverse effects of
BP on cardiovascular health, but an adequate and sustained control
of BP is achieved in only ;50% of hypertensive cases (2,3).
Therefore, nutritional and lifestyle-based interventions are unani-
mously recognized as important strategies for the primary preven-
tion of hypertension and as adjuvants in pharmacological therapies
to reduce cardiovascular risk (4). The Dietary Approach to Stop
1
Author disclosures: M. Siervo, J. Lara, I. Ogbonmwan, and J. C. Mathers, no
conflicts of interest.
2
Supplemental Figure 1–4 and Supplemental Tables 1–5 are available from the
‘‘Online Supporting Material’’ link in the online posting of the article and from the
same link in the online table of contents at http://jn.nutrition.org.
3
PROSPERO Database registration: CRD42012002144, http://www.crd.york.
ac.uk/prospero/.
4
Abbreviations used: BP, blood pressure; cGMP, cyclic GMP; DASH, Dietary
Approach to Stop Hypertension.
* To whom correspondence should be addressed. E-mail: mario.siervo@ncl.ac.uk.
818 Manuscript received September 28, 2012. Initial review completed November 3, 2012. Revision accepted February 28, 2013.
Hypertension (DASH) is one of the most effective nutritional
interventions for the prevention and nonpharmacological man-
agement of hypertension (5,6). The diet highlights the impor-
tance of an increased fruit and vegetable intake (7,8) and recent
research has suggested that the beneficial effects of the DASH
diet on BP are related to the high inorganic nitrate content of
some of the food products included in the DASH dietary plan
(e.g., green leafy and root vegetables) (9).
Larsen et al. (10) tested for the first time in a double-blind
crossover study the effects of sodium nitrate on BP in healthy
volunteers and reported a significant reduction in diastolic BP
(23.7 mm Hg). Beetroot is particularly rich in inorganic nitrate
content (typically ranging from 110 to 3670 mg nitrate
kg21) (11)
and it has therefore been utilized in several studies as a nutritional
strategy to test the effects of inorganic nitrate intake on BP. For
example, Webb et al. (12) showed in healthy participants that 24 h
after a single dose of 500 mL beetroot juice, systolic and diastolic
BP were reduced by 10.4 and 8.0 mm Hg, respectively.
The BP-lowering effects of inorganic nitrate may derive from
increased generation of nitric oxide (NO) (13,14), a pleiotro-
pic molecule involved in the vasodilation of large arteries and
resistance vessels (13,14). The endothelial isoform of the NO
synthase uses arginine and molecular oxygen as precursors to
ã 2013 American Society for Nutrition.
First published online April 17, 2013; doi:10.3945/jn.112.170233.
tonically release NO in the endothelium, which is important for the
control of vascular tone, smooth muscle growth, platelet aggrega-
tion, and inflammation (15,16). Reduced NO bioavailability has
been associated with impairment of endothelial function and
increased risk of hypertension and cardiovascular diseases (17–20).
A nonenzymatic pathway for the generation of NO in humans
has been proposed (nitrate-nitrite-NO pathway), which may con-
tribute to increased NO bioavailability (21). Dietary and endog-
enous inorganic nitrate molecules may be reduced by facultative
anaerobic bacteria on the dorsal surface of the tongue to nitrite,
which can be chemically (low pH) and enzymatically (xanthine
oxidoreductase, myoglobin, cytochrome P450, complexes of the
mitochondrial electron transport chain) further reduced to NO
(21). This pathway may represent a plausible mechanism to
explain the beneficial effects of dietary inorganic nitrate intake
on BP (21). The main mechanistic pathways involved in the
enzymatic and nonenzymatic generation of NO are described
in Supplemental Figure 1.
Here, we conducted a systematic review of the evidence from
randomized studies investigating the efficacy of inorganic nitrate
and beetroot supplementation on BP in humans. The results will
inform whether inorganic nitrate supplementation and nitrate-
enriched diets could be considered as effective nutritional strategies
for the prevention of hypertension and cardiovascular diseases.
Methods
The present systematic review was conducted according to estab-
lished guidelines and is reported according to PRISMA guidelines (22).
Types of studies. Randomized clinical trials in human participants
were included and the specific characteristics and designs of the trials
(type of placebo, parallel or crossover design, blinding of the interventions,
and duration) were assessed.
Subjects. Adult male and female participants (age >18 y) with or without
health comorbidities (hypertension, diabetes, and peripheral arterial
diseases) were included. Studies reporting data from participants with
different BMIs, ethnic backgrounds, and physical activity levels were
not excluded.
Types of interventions. Randomized clinical trials investigating the
effects of inorganic nitrate or beetroot juice supplementation and providing
information on the type of nitrate salt (potassium or sodium nitrate), volume,
formulation, frequency, and route of administration were included. Studies
that delivered the nitrate or beetroot supplementation alongside another
intervention (e.g., exercise, pharmacological agent, or dietary supple-
ment) were excluded if the interventions were different between groups. A
combined meta-analysis model was derived for inorganic nitrate solutions
and beetroot juice on BP. This approach was based on the evidence that
inorganic nitrate is rapidly absorbed from the stomach and proximal small
intestine with high bioavailability (23). A crossover study was specifically
conducted to determine the oral bioavailability of nitrate from spinach,
lettuce, and beetroot (24). A high bioavailability of the nitrate present in
the 3 food products (98 6 12% for spinach, 110 6 14% for lettuce, and
110 6 15% for beetroot) compared with nitrate in aqueous solution was
observed, which supports our analytical strategy to determine the pooled
effect of the 2 interventions (24).
Outcome measures. The primary outcomes of the analyses were changes
in diastolic and systolic BP after nitrate and/or beetroot supplementation. If
reported, changes in nitrate and nitrite concentrations in biological fluids
(plasma, urine, saliva) were also assessed.
Search strategy. A literature search of the PubMed, Embase, and
Scopus databases was undertaken from inception to February 2013. The
systematic review was restricted to articles published in English. The
search was conducted based on predefined search terms (dietary, inorganic,
nitrate, beetroot, beet root, BP, hypertension, vascular, NO, endothelial)
and using specific building blocks (Boolean terms, truncation) to create the
algorithms entered in each database. The full details of the algorithms are
reported in the Supplemental Methods (Box 1).
Selection of studies. Two investigators (M.S., I.O.) assessed articles
independently for eligibility. The first screening phase was based on the
analysis of titles and abstracts. When full agreement had been reached,
the article was either discarded or moved to the next phase. In case of
disagreement, the article was moved to the next phase to increase the
inclusiveness level. Reference lists of included papers and relevant reviews
were searched for articles potentially missed during the electronic search. In
the second phase, the full text of the selected articles was independently
assessed by 2 investigators. When full agreement had been reached, the
article was either discarded or moved to the next phase for full data
extraction. In case of disagreement, the article was evaluated by a third
Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
investigator (J.L.) and a final decision was reached by consensus.
Data extraction and bias. Two investigators extracted the data using a
standardized data collection form. A list of the extracted variables is
provided in the Supplemental Methods (Box 2). When BP measurements
were incomplete, the corresponding authors were contacted to request
the missing data. The quality of each study was assessed using the Cochrane
Risk of Bias Tool (25) to assess the following domains: selection bias
(random sequence generation and allocation concealment), performance
bias (blinding of participants and personnel to which intervention a
participant received), detection bias (blinding of outcome assessment),
attrition bias (completeness of outcome data, including attrition and
exclusions from the analysis), and reporting bias (selective outcome
reporting).
Measurement of treatment effect. The qualitative and quantitative
interpretation of the data was based on the absolute mean differences in
BP (in mm Hg) relative to baseline readings and SD of the mean differences
(SDdiff). For matched study design (crossover studies), the effect of dietary
supplementation (inorganic nitrate or beetroot juice) on BP was calculated
as the difference between the supplementation and placebo groups at the
end of each intervention. The SDdiff was calculated using the SDs for each
intervention (SDE = intervention; SDC = control) and an imputed
correlation coefficient (r) for the pre-post measurements (26). The
formula used for the calculation of the SDdiff is:
pffiffiffiffiffiffiffiffiffi
SDdiff ¼ SD2E þ SD2C 2ð23r3SDE 3SDC Þ:
An imputed correlation value of 0.50 was used to provide a conservative
estimate based on the assumption that this value would minimize the error
of the effect size estimates. A sensitivity analysis was conducted to test the
validity of the approach by entering a sequential range of low correlation
coefficients (r = 0.10, r = 0.25) to evaluate changes in the effect size of the
models.
Meta-analysis. A meta-analysis was conducted using Comprehensive
Meta-Analysis software (Biostat). Data are presented as mean differences
of systolic BP and diastolic BP (in mm Hg) and 95% CI. A random effect
model was used to provide a more conservative estimate of the pooled
effect size for diastolic and systolic BP. The BP differences were combined
across studies using a weighted DerSimonian-Laird random effects model
(27). Forest plots were generated for graphical presentations of the BP
outcomes. Statistical heterogeneity across studies was assessed using the I2
and the Q tests according to specific categories (low = 25%, moderate =
50%, high = 75%) and significance level (P < 0.10), respectively (28).
Funnel plots and EggerÔs regression test were used to evaluate potential
publication bias and selective reporting bias. Additional analyses were
conducted to evaluate the impact of publication bias on the effect size
by removing studies with the largest effects. Sensitivity analyses were
conducted to evaluate whether changes in diastolic and systolic BP were
influenced by study duration (<3 vs. $3 d), type of intervention (beetroot
juice vs. inorganic nitrate solution), and the removal of studies including
highly trained participants. One study was published in abstract form only
Inorganic nitrate, beetroot juice, and blood pressure 819
and reported a nonsignificant effect of inorganic nitrate on BP (29). This
study was not included in the meta-analysis, because full data were not
available after request. The potential influence of the nonsignificant results
from the study on the pooled systolic BP and diastolic BP estimates was
evaluated by entering into the model a null effect (mean difference = 0), the
mean of the pooled SD, and the sample size of each study. A random
effects meta-regression model was conducted to evaluate whether changes
in plasma nitrite concentrations (in nmol/L), study duration (in hours),
and daily dose of inorganic nitrate (in mmol) were associated with mean
differences in diastolic and systolic BP.
Results
Main search. A total of 10,184 articles was identified by the
primary search and, after the removal of duplicates (n = 6607),
3577 articles were screened for titles and abstracts. Then 41 articles
were selected for a full-text review and 16 studies (10,12,29–42)
were included in the systematic review (qualitative analysis) after
the exclusion of 25 articles. One study was published as abstracts
only (29). However, this study matched the inclusion and exclusion
criteria and it was included in the systematic review (qualitative
analysis) but excluded from the meta-analysis (quantitative anal-
ysis), because the full results on changes in BP were not available. A
flow chart of the literature search is shown in Figure 1. All eligible
studies used a crossover, double-blind, placebo-controlled, ran-
domized study design. The trials were conducted between 2006
and 2012 and included a total of 254 participants, with 7–30
participants per individual study. The duration of the interventions
ranged from 2 h to 15 d and the washout period from 6 to 28 d.
One article reported the results of 3 individual trials from which 2
trials were eligible and were entered in the final analysis (33). The
main characteristics of the studies included in the analysis are
presented in Supplemental Table 1.
FIGURE 1 Flow chart of the
literature search. BP, blood pres-
sure.
820 Siervo et al.
Participant characteristics. The majority of the studies recruited
young, nonsmoking, healthy participants and 2 studies were
conducted in older healthy participants (38) and overweight
and obese older individuals with type 2 diabetes (40). In addition,
most of the participants were men (;60%) and the male:female
ratio was not reported in 2 studies (33,42). The mean BMI of the
participants ranged between 22.3 and 30.8 kg
m22 and was not
reported in 2 studies (10,36). Seven studies investigated the effects
of inorganic nitrate and beetroot juice on exercise performance
and recruited primarily young, physically active men (30–
32,34,35,37,38).
Nitrate supplementation. Beetroot juice and nitrate salt
(sodium nitrate/potassium nitrate) supplementation was tested
in 12 (12,30–35,37–41) and 5 (10,29,33,36,42) studies, respectively.
The daily amount of inorganic nitrate (sodium or potassium nitrate)
Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
consumed in these studies ranged from ;2.5 to 24 mmol/dose
(157–1488 mg). Nitrate was dissolved either in distilled water
or low-nitrate water. The daily amount of nitrate in the beetroot
juice consumed varied between 5.1 and 45 mmol/dose (321–2790
mg). The volume of the beetroot juice drinks ranged from 140 to
500 mL/d and the beetroot juice was given as a concentrated
solution in 2 studies (32,38). The choice of the placebo varied
between studies and included equivalent volumes of water (3 studies)
(12,33,41), isomolar potassium and sodium chloride solutions
(5 studies) (10,29,33,36,42), blackcurrant juice (3 studies) (30,31,37),
apple juice (1 study) (39), and nitrate-depleted beetroot juice
(5 studies) (32,34,35,38,40). All solutions were given orally. Partic-
ipants were asked to refrain from consuming nitrate-enriched foods
in 9 studies (10,12,30,31,33,35,36,41–42), whereas participants
were invited to maintain their prestudy dietary habits and physical
activity level in 6 studies (32,34,37–40). The compliance with
the dietary prescriptions (low-nitrate diet, habitual dietary habits)
 TABLE 1 Meta-analysis of randomized clinical trials reporting the effects of beetroot juice and inorganic
nitrate supplementation on systolic and diastolic BP1
Effect size [mean
Studies difference (95% CI)]
n mm Hg
Systolic BP
,3 d 8 24.9 (27.6, 22.2)
$3 d 8 23.8 (25.6, 22.1)
Diastolic BP
,3 d 7 20.2 (21.3, +0.9)
$3 d 7 21.7 (23.6, +0.1)
Type of intervention
Systolic BP
Beetroot juice 12 24.5 (26.4, 22.5)
Inorganic nitrate 4 24.2 (26.1, 22.2)
Diastolic BP
Beetroot juice 10 20.9 (22.3, +0.5)
Inorganic nitrate 4 21.3 (23.0, +0.4)
1
The meta-analysis was stratified by study duration (,3 d, $3 d) and type of intervention (beetroot juice, inorganic nitrate). A random model
was applied to each subgroup to obtain the pooled estimate of the mean difference. Variability of the estimates was reported as 95% CI.
Heterogeneity of the studies in each subgroup of studies was evaluated using the Q test and the I2 test. BP, blood pressure.
was only evaluated in a few studies using diet diaries (37) of 24-h
dietary recall (34,35). The assessment of the compliance to the
nitrate supplementation was not reported, but the majority of the
studies measured plasma or urinary concentrations of nitrate and/
or nitrite, which could have provided information on the adher-
ence to the nitrate supplementation. One study did not report the
measurement of nitrate/nitrite concentrations (39).
Study quality. Resting BP was the primary outcome in 15 trials
(10,12,29–38,42) and ambulatory 24-h BP was used in 3 studies
(39–41). Three studies reported that the main aim of the study
was concealed (30,31,37) to the study participants (30,31,40).
All studies were double blind and reported information on the
duration of washout periods and dropout rates. The order of the
interventions in each trial was randomized. Three studies did not
report data on diastolic BP (29,34,35). Therefore, a meta-analysis
of the effects of inorganic nitrate and beetroot supplementation on
systolic and diastolic BP was performed for 16 (10,12,30–42) and
13 (10,12,30–33,36–42) studies, respectively. Funding sources
were disclosed in the majority of the studies, whereas 6 studies
declared potential conflicts of interest (33,35,38–41). The majority
of studies was rated high quality or low risk of bias. A small
proportion of studies were rated as having high performance
and detection bias (Supplemental Fig. 2).
Adverse events. Information on adverse events that occurred
during the study was reported in 8 studies (12,30,31,33,37,40–
42). Only one study reported a major adverse event (heartburn)
requiring the exclusion of the participant from the trial (33).
However, the unblinding of the study codes revealed that the
participant was assigned to the placebo intervention, which
excluded the link of nitrate with the onset of the event. The most
common side effect reported in the beetroot juice trials was
beeturia (red urine) and red stools (12,30,31,37,40–42).
BP qualitative results (systematic review). Twelve studies
showed a significant reduction in systolic BP (12,29–31,33–
35,37,38,41,42), whereas a significant change in diastolic BP
was observed in 6 studies (10,30,33,36–38). Beetroot juice supple-
mentation had a greater effect than inorganic nitrate on both
P value I2 Q value (P value)
,0.001 77.6 31.3 (,0.001)
,0.001 46.2 13.0 (0.07)
0.71 0 2.5 (0.86)
0.07 69.1 19.4 (0.003)
,0.001 73.7 41.4 (,0.001)
Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
,0.001 0.5 2.5 (0.47)
0.21 58.8 21.6 (0.01)
0.13 25.1 4.1 (0.26)
systolic and diastolic BP. The beneficial effects of beetroot juice
supplementation on BP were not confirmed when ambulatory
24-h BP monitoring was used to measure changes in BP (39–
41), including a group of older (>60 y) overweight and obese
participants with type 2 diabetes (40) (Supplemental Table 2).
BP quantitative results (meta-analysis). Inorganic nitrate
and beetroot juice consumption was associated with greater
changes in systolic BP [24.4 mm Hg (95% CI: 25.9, 22.8);
P < 0.001] (Fig. 2) than diastolic BP [21.1 mm Hg (95% CI:
22.2, 0.1); P = 0.06] (Fig. 3). The stratification of the analyses
by duration (<3 and $3 d) and type of interventions (beetroot
juice and inorganic nitrate salts) did not modify the effects on
systolic BP, because the meta-analysis models were significant
in studies of a shorter [<3 d, 24.9 mm Hg (95%CI: 27.6, 22.2);
P < 0.001] (12,33,34,36,39,41,42) and longer [$3 d, 23.8 mm
Hg (95% CI: 25.6, 22.1); P < 0.001] duration. Similarly, the
stratification by study duration did not modify the effect on diastolic
BP, because the model was not significant for both longer studies
[$3 d, 21.7 mm Hg (95% CI: 23.6, 0.1); P = 0.07] (10,30–
32,35,37) and shorter [<3 d, 20.2 mm Hg (95% CI: 21.3, 0.9);
P = 0.71] interventions (12,33,34,36,39,41,42). The effects of
beetroot juice supplementation [24.5 mm Hg (95% CI: 26.4,
22.5); P < 0.001] (12,30–35,37–41) and inorganic nitrate
[24.2 mm Hg (95% CI: 26.1, 22.2); P < 0.001] (10,33,36,42)
on systolic BP were both significant, whereas neither beetroot
juice nor inorganic nitrate supplementation was associated with
significant changes in diastolic BP (Table 1). The meta-regression
showed that mean differences in systolic BP were directly associ-
ated with the daily dose of inorganic nitrate (P < 0.05) (Fig. 4A) but
not with the study duration (P = 0.67) (Fig. 4B) or plasma nitrite
concentrations (P = 0.40) (Fig. 4C). Changes in diastolic BP were
not associated with the the daily dose of inorganic nitrate, study
duration or plasma nitrite concentrations (Fig. 5).
Publication bias and heterogeneity. Funnel plots for systolic
BP revealed an overall symmetric distribution of the studies
around the mean effect size, indicating the absence of publication
bias (Supplemental Figs. 3 and 4). EggerÕs regression test confirmed
the nonsignificant association between the mean differences and
Inorganic nitrate, beetroot juice, and blood pressure 821
 Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
FIGURE 2 Forest plot of randomized clinical trials investigating the effects of beetroot juice and inorganic nitrate supplementation on SBP. A
random model was applied to obtain the pooled mean differences in SBP. Data for SBP are presented as mean differences and 95% CI. Two
trials were reported in the same manuscript (33). BP, blood pressure; SBP, systolic blood pressure.

the SEs of each study (P = 0.47). However, we advocate for a
careful interpretation of the results in consideration of the small
number of participants, the short duration of the interventions,
and the over-representativeness of healthy participants. We
observed a high heterogeneity for systolic (I2 = 66%, Q = 44.3,
P < 0.001) and moderate heterogeneity for diastolic (I2 = 45%,
Q = 25.7, P < 0.01) BP meta-analysis models. The stratification of
the meta-analysis by study duration and type of intervention
helped to identify potential sources of heterogeneity for systolic BP,
because shorter duration (<3 d) and beetroot juice supplementation
were associated with greater heterogeneity (Table 1). In addition,
the exclusion of the study with the largest effect (12) and of studies
including physical activity outcomes (30,31,34,35,37,38) did not
modify the heterogeneity for both systolic (Supplemental Table 3)
and diastolic (Supplemental Table 4) BP.
Sensitivity analysis. Imputation of different correlation coeffi-
cients (r = 0.1, r = 0.25 instead of r = 0.5) entered for the calculation
of the effect size did not change the results for both systolic and
diastolic BP (Supplemental Table 5). Similarly, the inclusion of
a null mean difference in systolic and diastolic BP [to evaluate
the potential effect of the study (29) reporting a nonsignificant
effect on BP] reduced the mean difference in systolic BP by 0.6
mm Hg (from 4.4 to 3.8 mm Hg), but the pooled effect size
remained significant (P < 0.001) (Supplemental Table 3). The
inclusion of the study did not modify the results for diastolic
BP (P < 0.05) (Supplemental Table 4). The removal of the
studies investigating primarily the impact of inorganic nitrate
and beetroot juice on exercise performance (30–32,34–38) did
not change the results for systolic BP (23.9 mm Hg; P = 0.003)
(Supplemental Table 3) and diastolic BP (0.2 mm Hg; P = 0.70)
(Supplemental Table 4).
Discussion
Summary of main results. Inorganic nitrate and beetroot juice
supplementation were associated with a significant decrease in BP. The
pooled effect for the 2 interventions showed a reduction in systolic
BP of 4.4 mm Hg with a more modest decrease (21.1 mm Hg)
in diastolic BP. Beetroot juice was not associated with greater
changes in systolic BP than inorganic nitrate solutions (24.5 vs.
24.2 mm Hg, respectively). The meta-regression indicated that a
higher daily amount of the dose of inorganic nitrate may be
associated with greater reductions in systolic BP. Overall, studies

FIGURE 3 Forest plot of randomized clinical trials investigating the effects of beetroot juice and inorganic nitrate supplementation on DBP. A
random model was applied to obtain the pooled mean differences in DBP. Data for DBP are presented as mean differences and 95% CI. Two
trials were reported in the same manuscript (33). BP, blood pressure; DBP, diastolic blood pressure.

822 Siervo et al.


were characterized by a small sample size, short duration, and
over-representation of young, healthy men.
Study quality, bias, and applicability of evidence. The
overall quality of the studies was high. All studies were random-
ized and double-blind and the crossover study design of the trials
was justified by the type of interventions and outcomes. All studies
reported the duration of washout periods, which was adequate to
minimize the influence of carryover effects. The studies reported a
high compliance with the interventions, which may be explained
by the short duration of the trials. Participants recruited in the
studies were mostly healthy, normal-weight men and therefore the
results may not be applicable to a female population or to
individuals with cardiovascular or metabolic diseases. Kapil et al.
(33) have suggested a possible interaction with gender, as the
BP-lowering effect of inorganic nitrate was more marked in
men. However, we were unable to perform a sensitivity analysis to
Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
FIGURE 4 Meta-regression of moderators of SBP
after inorganic and beetroot juice supplementation.
(A) Meta-regression on the effects of the daily dose
of inorganic nitrate (in mmol) on mean differences in
SBP. Slope = 20.12, Q = 3.8, d.f. = 1, P , 0.05. (B)
Meta-regression on the effects of the study dura-
tion (in hours) on mean differences in SBP. Slope =
0.005, Q = 0.69, d.f. = 1, P = 0.40. (C) Meta-
regression on the effects of plasma nitrite concen-
trations (in nmol) on mean differences in SBP.
Slope = 0.004, Q = 0.02, d.f. = 1, P = 0.87. SBP,
systolic blood pressure.
assess the interaction with gender, which needs to be tested in
future studies. One study investigated the effects of beetroot juice
supplementation in older (>60 y) overweight and obese individuals
with type 2 diabetes and reported no significant effect of beetroot
juice on 24-h ambulatory BP. A beneficial effect of a 3-d beetroot
juice supplementation on resting systolic and diastolic BP was
observed in older, healthy participants. In addition, the duration of
the interventions was short and it is not known whether the
observed BP-lowering effects will be maintained in the long
term. These are critical factors to evaluate to understand if nitrate/
beetroot interventions may represent effective strategies for the
primary and/or secondary prevention of hypertension-related
cardiovascular diseases.
Potential biases in the review process. This study has several
potential limitations. The meta-analyses are based on retrospective
analytical inference, which may be affected by several factors, such
Inorganic nitrate, beetroot juice, and blood pressure 823
 Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
FIGURE 5 Meta-regression of moderators of
DBP after inorganic and beetroot juice supplemen-
tation. (A) Meta-regression on the effects of the
daily dose of inorganic nitrate (in mmol) on mean
differences in DBP. Slope = 0.03, Q = 0.33, d.f. = 1,
P = 0.56. (B) Meta-regression on the effects of the
study duration (in hours) on mean differences in
DBP. Slope = 20.002, Q = 0.30, d.f. = 1, P = 0.58.
(C) Meta-regression on the effects of plasma nitrite
concentrations (in nmol) on mean differences in
DBP. Slope = 20.002, Q = 0.85, d.f. = 1, P = 0.35.
DBP, diastolic blood pressure.

as the quality of the studies included, inclusiveness of the search
strategy to identify eligible studies to include in the meta-analysis,
assumptions on consistency of methodologies applied across the
different studies, inconsistency in reporting study results, limited
accessibility to individual study data, and inability to ascertain the
exact source of heterogeneity. In addition, we limited our search to
articles published in English and in peer-reviewed journals, which
may have increased the risk of publication bias. The clear
delineation of a priori inclusion and exclusion criteria and the
comprehensive search of 3 major electronic databases and refer-
ence lists are likely to have minimized bias and increased the
representativeness of the results. A robust selection procedure
was particularly important in the identification of studies explor-
ing relationships among inorganic nitrate, beetroot juice, and BP
control. However, all studies were characterized by a small sample
size and short duration and most of the studies recruited young,
healthy, nonsmoking men, making the representativeness of the
824 Siervo et al.
results debatable. In addition, some of the studies included
individuals with high physical activity levels (30–32,34,35,37), as
the primary aim of the studies was to test the effect of inorganic
nitrate on exercise performance. However, the exclusion of these
studies did not modify the magnitude of the lowering effect on
systolic BP. The analysis was also based on the imputation of pre-
postintervention correlation of BP changes and the sensitivity
analysis confirmed that the models for systolic BP and diastolic
BP were not affected by the inclusion of different correlation values.
Agreements and disagreements with previous results. Our
analyses showed that beetroot juice supplementation (24.5 mm
Hg) may not be associated with a greater reduction in systolic BP
compared with inorganic nitrate supplementation (24.2 mm Hg).
However, a careful interpretation of the results is needed until the
effects of the 2 interventions can be tested in a trial specifically
designed to test this hypothesis. The results seem to indicate
that the similar effects on BP may be accounted for by comparable
daily doses of inorganic nitrate, because the mean intake in the
beetroot and nitrate studies was similar (beetroot, 12.0 6 13.2
mmol/dose; inorganic nitrate, 15.5 6 9.2 mmol/dose) and the
presence in beetroot juice of other bioactive compounds (vitamin C,
magnesium, betaine, and flavonoids) (43–46) influencing vascular
physiology may provide minimal additive BP-lowering effects.
The results are comparable with other nutritional interventions
modifying the NO pathway, such as oral arginine supplementa-
tion. A recent meta-analysis of the effects of oral L-arginine
supplementation on BP reported a significant effect on systolic
BP (25.3 mm Hg) and diastolic BP (22.6 mm Hg) (47), which are
comparable with the effect sizes observed in our meta-analysis.
Plasma nitrite concentrations are a biomarker of NO bioavail-
ability (13). However, our analyses showed a nonsignificant
association of plasma nitrite with changes in BP, which could
be related to the study heterogeneity (study duration, dosing
schedule, and amount of nitrate). These factors may have influenced
the pharmacokinetic profile of plasma nitrate and nitrite and altered
the association with BP. Two studies found a significant association
between changes in plasma nitrite concentrations and changes in
systolic BP after oral inorganic nitrate supplementation (12,33),
which supports our hypothesis on the lack of standardization of the
measurement protocols. Cyclic GMP (cGMP) is the second
messenger of the NO pathway and it represents a biomarker of
NO bioactivity (13). Only 2 studies have tested the relation-
ship between cGMP and changes in BP after oral inorganic nitrate
and beetroot juice supplementation. Both studies showed a greater
plasma cGMP concentration after ingestion of inorganic nitrate
and beetroot juice (33).
Implications for research practice and main conclusions.
Our results showed a significant decline in systolic BP after short-
term inorganic nitrate and beetroot juice supplementation, which
may potentially have important implications for the primary and
secondary prevention of cardiovascular diseases. The seventh
report of the Joint National Committee on BP estimated that a
systolic BP reduction of at least 5 mm Hg (as observed here) could
decrease the risk of mortality due to stroke by 14% and mortality
from cardiovascular diseases by 9% (48). However, before such
nutritional approaches can be recommended, the long-term
efficacy of inorganic nitrate needs to be tested in people at higher
cardiovascular risk together with an assessment of the tolerance,
safety, compliance, and cost-effectiveness of the interventions.
Acknowledgments
The authors are very grateful to Dr. Larsen, Professor Weitzberg,
Dr. Kapil, Professor Ahluwalia, Professor Lovegrove, Dr. Cermak,
and Professor Jones for providing the original data from their
studies. The systematic review was conceived by J.C.M., J.L.,
and M.S; I.O. and M.S. searched, collected, and analyzed the
data and co-wrote the manuscript; all authors contributed to
subsequent analyses and interpretation; all authors contributed
to the final revision of the manuscript; and M.S. is the guarantor
for the manuscript and had full access to all of the data in the
study and takes responsibility for the integrity of the data and
the accuracy of the data analysis. All authors read and approved
the final manuscript.
Literature Cited
1. Ezzati M, Lopez AD, Rodgers A, Vander Hoorn S, Murray CJ. Selected
major risk factors and global and regional burden of disease. Lancet.
2002;360:1347–60.
2. Cutler JA, Sorlie PD, Wolz M, Thom T, Fields LE, Roccella EJ. Trends
in hypertension prevalence, awareness, treatment, and control rates in
united states adults between 1988–1994 and 1999–2004. Hypertension.
2008;52:818–27.
3. Egan BM Zhao Y, Axon RN. US trends in prevalence, awareness,
treatment, and control of hypertension, 1988–2008. JAMA. 2010;303:
2043–50.
4. Savica V, Bellinghieri G, Kopple JD. The effect of nutrition on blood
pressure. Annu Rev Nutr. 2010;30:365–401.
5. Appel LJ, Brands MW, Daniels SR, Karanja N, Elmer PJ, Sacks FM.
Dietary approaches to prevent and treat hypertension: a scientific
statement from the American Heart Association. Hypertension.
2006;47:296–308.
6. Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks
FM, Bray GA, Vogt TM, Cutler JA, et al. A clinical trial of the effects of
dietary patterns on blood pressure. N Engl J Med. 1997;336:1117–24.
7. Moore TJ, Conlin PR, Ard J, Svetkey LP. DASH (Dietary Approaches to
Stop Hypertension) diet is effective treatment for stage 1 isolated
Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
systolic hypertension. Hypertension. 2001;38:155–8.
8. Al-Solaiman Y, Jesri A, Mountford WK, Lackland DT, Zhao Y, Egan
BM. DASH lowers blood pressure in obese hypertensives beyond
potassium, magnesium and fibre. J Hum Hypertens. 2010;24:237–46.
9. Hord NG, Tang Y, Bryan NS. Food sources of nitrates and nitrites: the
physiologic context for potential health benefits. Am J Clin Nutr.
2009;90:1–10.
10. Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of
dietary nitrate on blood pressure in healthy volunteers. N Engl J Med.
2006;355:2792–3.
11. European FSAE. Opinion of the Scientific Panel on Contaminants in the
Food chain on a request from the European Commission to perform a
scientific risk assessment on nitrate in vegetables. EFSA J. 2008;689:1–79.
12. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S,
Rashid R, Miall P, Deanfield J, et al. Acute blood pressure lowering,
vasoprotective, and antiplatelet properties of dietary nitrate via bioconver-
sion to nitrite. Hypertension. 2008;51:784–90.
13. Siervo M, Stephan BC, Feelisch M, Bluck LJ. Measurement of in vivo nitric
oxide synthesis in humans using stable isotopic methods: a systematic
review. Free Radic Biol Med. 2011;51:795–804.
14. Kelm M. Nitric oxide metabolism and breakdown. Biochim Biophys
Acta. 1999;1411:273–89.
15. Umans JG, Levi R. Nitric oxide in the regulation of blood flow and
arterial pressure. Annu Rev Physiol. 1995;57:771–90.
16. Bruckdorfer R. The basics about nitric oxide. Mol Aspects Med.
2005;26:3–31.
17. Avogaro A, de Kreutzenberg SV. Mechanisms of endothelial dysfunction
in obesity. Clin Chim Acta. 2005;360:9–26.
18. Avogaro A, de Kreutzenberg SV, Fadini G. Endothelial dysfunction:
causes and consequences in patients with diabetes mellitus. Diabetes Res
Clin Pract. 2008;82 Suppl 2:S94–101.
19. Siervo M, Corander M, Stranges S, Bluck L. Post-challenge hyper-
glycaemia, nitric oxide production and endothelial dysfunction: the
putative role of asymmetric dimethylarginine (ADMA). Nutr Metab
Cardiovasc Dis. 2011;21:1–10.
20. Charakida M, Deanfield JE, Halcox JPJ. The role of nitric oxide in early
atherosclerosis. Eur J Clin Pharmacol. 2006;62:69–78.
21. Lundberg JO, Gladwin MT, Ahluwalia A, Benjamin N, Bryan NS,
Butler A, Cabrales P, Fago A, Feelisch M, et al. Nitrate and nitrite in
biology, nutrition and therapeutics. Nat Chem Biol. 2009;5:865–9.
22. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gotzsche PC, Ioannidis
JP, Clarke M, Devereaux PJ, Kleijnen J, Moher D. The PRISMA
statement for reporting systematic reviews and meta-analyses of studies
that evaluate healthcare interventions: explanation and elaboration.
BMJ. 2009;339:b2700.
23. Hord NG. Dietary nitrates, nitrites, and cardiovascular disease. Curr
Atheroscler Rep. 2011;13:484–92.
24. Lambers A, Kortboyer, JM, Schothorst, RC, Sips, AJAM, Cleven RFMJ,
Meulenbelt J. The oral bioavailability study of nitrate from vegetables
in healthy volunteers. Bilthoven (Netherlands): National Institute of
Public Health and the Environment; 2000.
25. Higgins JPT, Altman DG, Gøtzsche PC, Jüni P, Moher D, Oxman AD,
Savović J, Schulz KF, Weeks L, Sterne JAC. The Cochrane CollaborationÕs
tool for assessing risk of bias in randomised trials. BMJ. 2011;343:
d5928.
Inorganic nitrate, beetroot juice, and blood pressure 825
26. Higgins J, Green S. Cochrane handbook for systematic reviews of
interventions version 5.1.0. The Cochrane Collaboration; 2011 [updated
March 2011] [cited 2013 Jan 23]. Available from: www.cochrane-
handbook.org.
27. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin
Trials. 1986;7:177–88.
28. Higgins JPT, Thompson SG, Deeks JJ, Altman DG. Measuring
inconsistency in meta-analyses. BMJ. 2003;327:557–60.
29. Lidder S, Hunt J, Omar S, Webb A. Acute effects of dietary nitrate on
glucose handling and insulin levels during an oral glucose tolerance test
in healthy subjects. Nitric Oxide -. Biol Chem. 2011;24:S32.
30. Bailey SJ, Fulford J, Vanhatalo A, Winyard PG, Blackwell JR, DiMenna
FJ, Wilkerson DP, Benjamin N, Jones AM. Dietary nitrate supplemen-
tation enhances muscle contractile efficiency during knee-extensor
exercise in humans. J Appl Physiol. 2010;109:135–48.
31. Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson
DP, Tarr J, Benjamin N, Jones AM. Dietary nitrate supplementation
reduces the O2 cost of low-intensity exercise and enhances tolerance to
high-intensity exercise in humans. J Appl Physiol. 2009;107:1144–55.
32. Cermak NM, Gibala MJ, Van Loon LJC. Nitrate supplementation’s
improvement of 10-km time-trial performance in trained cyclists. Int J
Sport Nutr Exerc Metab. 2012;22:64–71.
33. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F,
Rehman F, Arghandawi S, Pearl V, Benjamin N, et al. Inorganic nitrate
supplementation lowers blood pressure in humans: role for nitrite-derived
NO. Hypertension. 2010;56:274–81.
34. Lansley KE, Winyard PG, Bailey SJ, Vanhatalo A, Wilkerson DP,
Blackwell JR, Gilchrist M, Benjamin N, Jones AM. Acute dietary nitrate
supplementation improves cycling time trial performance. Med Sci
Sports Exerc. 2011;43:1125–31.
35. Lansley KE, Winyard PG, Fulford J, Vanhatalo A, Bailey SJ, Blackwell
JR, DiMenna FJ, Gilchrist M, Benjamin N, Jones AM. Dietary nitrate
supplementation reduces the O2 cost of walking and running: a
placebo-controlled study. J Appl Physiol. 2011;110:591–600.
36. Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Dietary nitrate
reduces maximal oxygen consumption while maintaining work perfor-
mance in maximal exercise. Free Radic Biol Med. 2010;48:342–7.
37. Vanhatalo A, Bailey SJ, Blackwell JR, DiMenna FJ, Pavey TG,
Wilkerson DP, Benjamin N, Winyard PG, Jones AM. Acute and chronic
effects of dietary nitrate supplementation on blood pressure and the
826 Siervo et al.
physiological responses to moderate-intensity and incremental exercise.
Am J Physiol Regul Integr Comp Physiol. 2010;299:R1121–31.
38. Kelly J, Fulford J, Vanhatalo A, Blackwell JR, French O, Bailey SJ,
Gilchrist M, Winyard PG, Jones AM. Effects of short-term dietary
nitrate supplementation on blood pressure, O2 uptake kinetics, and
muscle and cognitive function in older adults. Am J Physiol Regul
Integr Comp Physiol. 2013;304:R73–83.
39. Coles LT, Clifton PM. Effect of beetroot juice on lowering blood
pressure in free-living, disease-free adults: a randomized, placebo-
controlled trial. Nutr J. 2012;11:106–11.
40. Gilchrist M, Winyard PG, Aizawa K, Anning C, Shore A, Benjamin N.
Effect of dietary nitrate on blood pressure, endothelial function, and
insulin sensitivity in type 2 diabetes. Free Radic Biol Med. Epub 2013
Feb 8.
41. Hobbs DA, Kaffa N, George TW, Methven L, Lovegrove JA. Blood
pressure-lowering effects of beetroot juice and novel beetroot-enriched
bread products in normotensive male subjects. Br J Nutr. 2012;108:
2066–74.
Downloaded from https://academic.oup.com/jn/article-abstract/143/6/818/4571708 by guest on 03 May 2019
42. Bahra M, Kapil V, Pearl V, Ghosh S, Ahluwalia A. Inorganic nitrate
ingestion improves vascular compliance but does not alter flow-
mediated dilatation in healthy volunteers. Nitric Oxide. 2012;26:
197–202.
43. Cao G, Sofic E, Prior RL. Antioxidant capacity of tea and common
vegetables. J Agric Food Chem. 1996;44:3426–31.
44. Kaur C, Kapoor HC. Anti-oxidant activity and total phenolic content of
some Asian vegetables. Int J Food Sci Technol. 2002;37:153–61.
45. Pedreño MA, Escribano J. Studying the oxidation and the antiradical
activity of betalain from beetroot. J Biol Educ. 2000;35:49–51.
46. Georgiev VG, Weber J, Kneschke E-M, Denev P, Bley T, Pavlov A.
Antioxidant activity and phenolic content of betalain extracts from
intact plants and hairy root cultures of the red beetroot cv. Beta vulgaris
cv. Detroit Dark Red. Plant Foods Hum Nutr. 2010;65:105–11.
47. Dong J-Y, Qin L-Q, Zhang Z, Zhao Y, Wang J, Arigoni F, Zhang W.
Effect of oral l-arginine supplementation on blood pressure: a meta-
analysis of randomized, double-blind, placebo-controlled trials. Am
Heart J. 2011;162:959–65.
48. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL,
Jones DW, Materson BJ, Oparil S, et al. Seventh Report of the Joint
National Committee on Prevention, Detection, Evaluation, and Treat-
ment of High Blood Pressure. Hypertension. 2003;42:1206–52.