Journal of Strength and Conditioning Research

Effects of Nitrate Supplementation on Muscle Strength and Mass: A Systematic

Review
--Manuscript Draft--

Manuscript Number: JSCR-08-16096R4

Full Title: Effects of Nitrate Supplementation on Muscle Strength and Mass: A Systematic
Review

Short Title: Nitrate Supplementation on Muscle Strength and Mass

Article Type: Brief Review

Keywords: nitric oxide; beetroot juice; resistance exercise; Sport Performance; muscle protein
synthesis; ergogenic

Corresponding Author: Helios Pareja-Galeano, Ph.D.

Universidad Europea de Madrid
Villaviciosa de Odón, Madrid SPAIN

Corresponding Author Secondary

Information:

Corresponding Author's Institution: Universidad Europea de Madrid

Corresponding Author's Secondary

Institution:

First Author: Olivia K Anderson, MSc

First Author Secondary Information:

Order of Authors: Olivia K Anderson, MSc

Maria Martinez-Ferran, MSc

Jorge Lorenzo-Calvo, PhD

Sergio L Jiménez, PhD

Helios Pareja-Galeano, Ph.D.

Order of Authors Secondary Information:

Manuscript Region of Origin: SPAIN

Abstract: This systematic review examines the effect of dietary nitrate supplementation (SUP) on
muscle strength and hypertrophy when combined with physical exercise. The
databases PubMed, Web of Science, and Medline were searched for full-text articles
published between January 2000 and June 2020. For inclusion, studies had to report
on the effects of SUP administered as acute or chronic doses together with a
standardized exercise protocol on muscle strength and hypertrophy compared to
placebo in healthy adults who were sedentary, physically active, or professional
athletes. Twelve studies (1,571 participants) were finally selected. In five studies, the
SUP regime was acute and in seven it was chronic. SUP was nitrate-rich beetroot juice
in nine studies, a potassium nitrate capsule in one, and increased dietary nitrate in two.
Ingested nitrate was 64 to 1200 mg. Of the 12 studies, six observed an ergogenic
effect of SUP compared to placebo. These findings indicate that muscle strength gains
are possible provided the dose, format, frequency, period, and exercise test are
appropriate. Best results were observed for a minimum acute dose of 400 mg of nitrate
provided as beetroot juice/shot taken 2 to 2.5 h prior to exercise involving low and high
intensity muscle contractions. This SUP regime seems to improve muscle efficiency in
terms of reduced phosphocreatine and energy costs (p-magnetic resonance
spectroscopy), and improved time to exhaustion.

Response to Reviewers: Reviewer #1: Thank you to the authors for attending to the reviewer
comments/suggestions throughout this process. I believe the work you have done to
address the concerns have improved the manuscript.
Response to reviewer 1: We thanks this reviewer for his/her previous valuable revision.

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Reviewer #3: The authors have made changes to the manuscript, however there are
sections that were not properly addressed.
Response to reviewer 3: Thank you for your comments. We feel the comments made
have certainly helped us improve our work.

Point 1: Figure 2. Colors were removed, however there is not much contrast between
the light gray and dark gray bars. Make the contrast greater, and thus make it easier
for the reader to distinguish the differences in bias, by using black and white - closed
black bar and white bar with black outline.
Response point 1: We assume this reviewer refers to Figure 2. We have changed the
colors to black and white to make it easier to read

Point 2: Figure 3. Similar to that of figure 3, the colors were removed however the gray
circles make it more difficult to read. The circles should be removed. The use of + and
? are sufficient.
Response point 2: We assume this reviewer refers to Table 1. We have modified icons
as you suggested.

Point 3: Table 2. This table was very slightly changed. It is still far too busy to be
useful. Consider what is essential to be presented and revise. The columns run
together making it difficult to read. In the previous review comments, it was suggested
that the authors look to articles published in JSCR for examples. I am not sure that this
was done. Here are a few to view:
* Borja et al., JSCR 33(10): 2875-2881, 2019. https://journals.lww.com/nsca-
jscr/Fulltext/2019/10000/Swim_Specific_Resistance_Training__A_Systematic.31.aspx
* Simone et al., 33(10): 2882-2896, 2019. https://journals.lww.com/nsca-
jscr/Fulltext/2019/10000/Effects_of_Judo_Training_on_Bones__A_Systematic.32.aspx
* Baz-Valle, et al., 35(3): 870-878, 2021. https://journals.lww.com/nsca-
jscr/Fulltext/2021/03000/Total_Number_of_Sets_as_a_Training_Volume.39.aspx
Response point 3: We appreciate your feedback and have modified the table
accordingly. We have divided the columns to make the table easier to read. Also, we
have deleted the columns “Other variables” and “Muscle hypertrophy and strength
variables” to release expendable content and the order of the studies has been
changed to show acute designs first, followed by the chronic designs and finally those
studies that combine chronic and acute designs. Finally, we have considerably
reduced the content by summarizing. The authors are convinced that the table has
improved substantially.

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Title Page (Showing Author Information)

TITLE: Effects of Nitrate Supplementation on Muscle Strength and Mass:

A Systematic Review

AUTHORS: Olivia K. Anderson1, María Martinez-Ferran1, Jorge Lorenzo-

Calvo2, Sergio L. Jiménez3, Helios Pareja-Galeano4

Affiliations:

1
Faculty of Sport Sciences. Universidad Europea de Madrid. Madrid, Spain.

2
Department of Sports, Universidad Politécnica de Madrid, Facultad de Ciencias de la

Actividad Física y del Deporte. Madrid, Spain.

3
Centre for Sport Studies. Universidad Rey Juan Carlos. Madrid, Spain.

4
Department of Physical Education, Sport and Human Movement, Autonomous University of

Madrid, Madrid, Spain

Running head: Nitrate Supplementation on Muscle Strength and Mass

Funding: Helios Pareja-Galeano is supported by a grant from Universidad Europea de Madrid

(#2019/UEM01).

Corresponding Author:
Dr. Helios Pareja-Galeano
Department of Physical Education, Sport and Human Movement.
Autonomous University of Madrid.
Av. Francisco Tomás y Valiente 3, Madrid, Spain 28049.
Email: helios.pareja@uam.es
Manuscript ( NO AUTHOR INFORMATION - Manuscript Text
Pages, including References and Figure Legends)

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TITLE: Effects of Nitrate Supplementation on Muscle Strength and Mass:
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7 A Systematic Review
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 1 2
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4 ABSTRACT
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7 This systematic review examines the effect of dietary nitrate supplementation (SUP) on muscle
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9 strength and hypertrophy when combined with physical exercise. The databases PubMed, Web of
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11 Science, and Medline were searched for full-text articles published between January 2000 and June
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14 2020. For inclusion, studies had to report on the effects of SUP administered as acute or chronic
15
16 doses together with a standardized exercise protocol on muscle strength and hypertrophy compared
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18 to placebo in healthy adults who were sedentary, physically active, or professional athletes. Twelve
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20 studies (1,571 participants) were finally selected. In five studies, the SUP regime was acute and in
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23 seven it was chronic. SUP was nitrate-rich beetroot juice in nine studies, a potassium nitrate capsule
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25 in one, and increased dietary nitrate in two. Ingested nitrate was 64 to 1200 mg. Of the 12 studies,
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27 six observed an ergogenic effect of SUP compared to placebo. These findings indicate that muscle
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29 strength gains are possible provided the dose, format, frequency, period, and exercise test are
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appropriate. Best results were observed with a minimum acute dose of 400 mg of nitrate provided
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34 as beetroot juice/shot taken 2 to 2.5 h prior to exercise involving low and high intensity muscle
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36 contractions. This SUP regime seems to improve muscle efficiency in terms of reduced
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38 phosphocreatine and energy costs (p-magnetic resonance spectroscopy), and improved time to
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exhaustion.
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46 Keywords: nitric oxide, beetroot juice, resistance exercise, sport performance, muscle protein
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48 synthesis, ergogenic
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4 1. INTRODUCTION
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7 In high-level competition, athletes often look to nutritional supplements to enhance their
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9 performance. An improvement of 0.6% could make the difference between Olympic medalists and
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11 non-medalists (33). The global dietary supplement market size was estimated at USD 123.3 billion
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14 in 2019 and is expected to reach USD 132.3 billion in 2020 (11). This field is constantly growing
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16 and evolving with hundreds of research papers published annually (14). In the year 2012 alone,
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18 2,082 articles were published under the key worlds “sports nutrition” (14).
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21 The International Society of Sports Nutrition (ISSN) published an updated review article in 2018
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23 presenting an overview of the science related to optimization of training and performance
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25 enhancement through exercise training and nutrition. According to this review, a limited number of
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28 supplements, including nitrates, are categorized as having “strong evidence to support efficacy and
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30 apparently safe”(14).
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33 Inorganic nitrates have the ability to promote pathways leading to the production of nitric oxide
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35 (NO). This is a relatively recent finding, as NO was believed to be generated solely through the
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37 oxidation of L-arginine in a reaction catalyzed by a group of NO synthase (NOS) enzymes,
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39 resulting in the production of nitrate and nitrite (1,26). NO is a signaling molecule that induces
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smooth muscle relaxation and takes part in processes that could enhance muscle contraction
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44 (21,31,32).
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Beetroot juice is used as a supplement because it may serve as a precursor of nitric oxide, due to its
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49 high nitrate content (1). Numerous studies investigated beetroot juice and its effects on intensive
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51 endurance exercise modalities, and concluded it does in fact enhance performance when the
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53 dominant type of energy metabolism is oxidative (3,13,17,19,26). Additionally, there is reason to
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believe that enhanced NO bioavailability may influence muscle function and exercise performance
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58 by modulating skeletal muscle function through its role in the regulation of blood flow,
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60 contractility, and glucose and calcium homeostasis (1,21).
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4 In this review, we address the limited but growing amount of research that investigated the possible
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6 ergogenic effects of beetroot supplementation (SUP) on muscle strength and hypertrophy gains
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9 produced in response to resistance training. Accordingly, this systematic review examines the
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11 effects of oral nitrate SUP, both acute and chronic, on muscle strength and hypertrophy when
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13 combined with resistance exercise.
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19 2. METHODS
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21 This systematic review was written in accordance with the Preferred Reporting Items for Systematic
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24 Reviews and Meta-Analyses (PRISMA) recommendations (20).
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2.1 Eligibility criteria
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29 2.1.1 Inclusion criteria
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32 We adhered to the eligibility criteria of the PICOS (population, intervention, comparators, outcomes
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34 and study design) model (34). The population examined was composed of healthy male and female
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36 subjects of any age who were sedentary, physically active, or professional athletes. The
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39 interventions considered were those in which a nitrate supplement was administered either acutely
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41 or chronically with standardized exercise. The comparators were effects observed between placebo
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43 and nitrate SUP. Outcomes observed were those relating the use of nitrate SUP with muscle
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45 strength and hypertrophy. Finally, it was required that the design of the studies included was at least
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48 single blind and randomized.
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50 2.1.2 Exclusion criteria
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53 The following studies were excluded: observational (cohort, case-control, ecological, case reports,
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55 and case series); animal; in vitro conducted on cells or tissues; those conducted in injured or ill
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58 participants; those not including standardized physical exercise; non-blind; and those in which
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4 nitrate SUP was administered with other supplements. Also excluded were articles with no full-text
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6 available, opinion pieces, review articles, commentaries and editorials.
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9 2.2 Literature Search
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12 Studies were identified by searching the databases PubMed, Web of Science, Medline, CINAHL,
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14 and SPORT Discus. The search terms used were: (nitrate OR beetroot) AND ("skeletal muscle" OR
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16 "muscle mass" OR "muscle protein synthesis" OR "hypertrophy" OR "strength" OR “resistance”)
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19 AND (supplementation).
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2.3 Study Selection
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24 Once the records were identified, duplicates were first removed. Next, based on the titles and
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26 abstracts of articles, all those that did not meet eligibility criteria were excluded. Finally, the full
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29 texts of the resulting articles were read, and those that did not meet the inclusion criteria were
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31 excluded.
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34 2.4 Data Extraction
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36 The information extracted from the records identified was: authors and year of publication;
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39 participant characteristics (level of activity or sports discipline, number of subjects, age, gender, and
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41 health status); and intervention protocol (nitrate source, dose, period of SUP, type of exercise). The
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43 main outcomes recorded were: maximal voluntary torque (MVT), maximal voluntary contraction
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45 (MVC), muscle strength, muscle hypertrophy, and exercise performance.
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48 2.5 Quality Assessment and Risk of Bias
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51 The quality of each investigation was assessed in accordance with the Cochrane Collaboration
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53 Guidelines (9). The Cochrane Risk of Bias tool for randomized clinical trials is based on seven
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55 domains: sequence generation and allocation concealment (selection bias), blinding of participants
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58 and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete
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4 outcome data (attrition bias), selective reporting (reporting bias) and other sources of bias (reporting
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6 bias). Risk of bias was categorized as low, high or unclear.
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12 3. RESULTS
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15 3.1 Study Selection
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Through the database searches, 862 records were identified. Five additional studies were identified
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20 in a manual search. Of these records, 457 duplicates were removed and 418 articles excluded after
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22 their titles and abstracts were screened for eligibility. This left 39 studies, which were assessed for
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24 eligibility. Of these, another 27 were removed according to our inclusion/exclusion criteria. The
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26 studies finally included were 12 randomized placebo-controlled trials (Figure 1).
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29 [Figure 1 about here]
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32 3.2 Characteristics of the Studies
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35 First, we determined the main characteristics of the 12 studies selected for this systematic review.
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37 The total number of participants was 1571: 136 men and 1435 women. The majority of the studies
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39 had less than 25 participants, and only two of the 12 studies assessed the effects of nitrate in a larger
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41 group of subjects (n=1420 and n=35) (18,30). All study participants had a good overall health
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44 status. However, physical activity level varied among individuals. Thus, seven studies worked with
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46 moderately trained or recreationally active individuals (7,10,12,18,27,29,38) and only one study had
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48 participants categorized as healthy, not specifying their level of physical activity (30). The
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50 remaining four studies included athletes of specific modalities such as combat sport (n=1) (23),
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53 CrossFit (n=1) (16), and resistance trained (n=2) (28,38). While most of the study participants were
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55 aged 20 to 30 years, in one study they were 75.2 ± 2.7 years old (30). The SUP design of the studies
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57 reviewed was classified as either acute (5 studies) or chronic (7 studies). In the former studies, the
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59 acute nitrate dose was administered 120 min to 4 h before the exercise test (18,24,28,29,38). In the
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4 seven studies examining the effects of chronic SUP, nitrate doses were given from 4 to 15 days
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6 prior to exercise testing (7,10,12,16,22,27,30). The nitrate source was beetroot juice or gel in nine
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9 studies (7,10,12,18,22,24,28,29,38), a nitrate capsule in one study (16), and increased total nitrate
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11 intake through diet manipulation in two studies (27,30). The doses given in the studies varied based
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13 on the SUP protocol. For beetroot juice or gel, the dose varied from 400 to 1200 mg, the capsule
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15 formulation contained 250 mg per capsule (500 mg per day), and to increase nitrate intake through
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18 diet, the total amount given varied from 64 mg to 525 mg per day. Two studies utilized a Food
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20 Frequency Questionnaire (FFQ) (18,30) and had subjects report on their dietary habits. Four studies
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22 worked with 24 h (28), 48 h (10), 72 h (16) or one-week (27) diet recalls. In another four studies,
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24 participants were only instructed to follow their normal diet for either the entirety of the study (7) or
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the day preceding exercise testing (22,29,38).
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29 3.3 Quality Assessment and Risk of Bias
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32 All 12 studies were categorized as low risk of bias in the “random sequence generation” category.
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34 In the allocation concealment category, seven studies were considered as low risk
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(10,12,18,22,28,29,38), but five different studies were unclear (7,16,24,27,30). With regards to
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39 performance, ten studies (double-blind) were defined as low risk (7,10,12,16,18,22,24,28,29,38),
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41 and two studies (not defined) (27,30) fell under unclear risk. In the blindness of outcome
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43 assessment, only one study was considered low risk (16), while 11 studies were unclear
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(7,10,12,18,22,24,27–30,38). For the category “incomplete outcome data”, all studies were low risk.
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48 For selective outcome reporting, all studies were considered to be low risk. Lastly, ten studies
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50 showed an overall low risk of bias (7,10,12,16,18,22,24,28,29,38), and two were considered to be of
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52 unclear risk (27,30), while none were determined to have a high risk of bias. This information is
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54 detailed in Table 1 and Figure 2.
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57 [Table 1 about here]
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7 3.3 Results of Individual Studies
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10 The studies selected for this systematic review were categorized by duration of nitrate SUP and
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12 exercise protocol. In five studies SUP was acute and the dose of nitrate was administered 120 min
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14 to 4 h before the exercise test (18,24,28,29,38). In the other seven studies (chronic SUP), an acute
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16 exercise protocol was completed as either a single bout of exercise (10,12,16,22,27,30) or a few
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19 exercise sessions per week or month (7) (Table 2).
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21 [Table 2 about here]
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24 3.3.1 Acute Supplementation with Nitrate
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27 De Oliveira et al. (24) reported on the SUP of 14 men who practiced recreational combat sports
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29 with either 100 g of beetroot gel containing 756 mg nitrate or a nitrate-depleted gel 120 min before
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31 exercise. The subjects performed three sets of rhythmic handgrip exercise: MVC of the forearm
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34 muscles was measured before and 20 min after performing the handgrip test. These researchers
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36 noted that a single dose of nitrate SUP attenuated the decline in handgrip strength by improving
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38 ∆MVC after three bouts of exhaustive handgrip exercise (24). Additionally, Rodríguez-Fernández
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40 et al. (29) supplemented 18 active males with 800 mg of nitrate in the form of two 70 mL bottles of
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43 Beet It Sport® or two 70 mL bottles of nitrate depleted PLA 2.5 h prior to testing. Participants
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45 performed four sets of eight maximal half-squat repetitions, each set completed against different
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47 inertial loads, and separated by 3 min of passive recovery. Compared to PLA, SUP resulted in
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49 increased total mean power at all moment inertias analyzed (29). Williams et al. (38) recruited 11
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resistance trained men and supplemented them with Beet It Sport®, but only used one 70 mL bottle
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54 containing 400 mg nitrate for SUP and one 70 mL bottle of identical blackcurrant juice for PLA
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56 (with a negligible nitrate content) 2 h before exercise testing. Subjects performed the barbell bench
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58 press with two sets of two repetitions at 70% of the one repetition maximum (1RM), separated by 3
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min of rest. After 5 min of rest, subjects completed three sets of repetitions until failure at the same
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4 load, each set separated by 2 min of rest. For total repetitions completed, numbers were
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6 significantly higher for the SUP than PLA condition. Mean power (watts) was significantly
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9 increased for SUP vs. PLA (38). Similarly, Ranchal-Sanchez et al. (28) supplemented 12 male
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11 participants with either 70 mL of Beet It Sport® containing 400 mg of nitrate or 70 mL of nitrate-
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13 depleted blackcurrant juice 120 minutes before exercise testing. The subjects performed back squats
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15 followed by bench press, with a 3-min rest interval between the two activities, and divided into
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18 three sets with load volumes of 60%, 70%, and 80% 1RM, respectively. These were carried out
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20 incrementally and with 3 min of rest between each set. Interestingly, significant differences were
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22 found in the total number repetitions of back-squat exercise, both conducted at 60% 1RM and at
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24 70% 1RM, while no differences were obtained at 80% 1RM in favor of SUP; and no differences
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were observed in the bench press exercise for any percentage of intensity. Lastly, Lee et al. (18)
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29 recruited 35 male and female recreationally active adults. Participants were supplemented with
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31 either two 70 mL “Beet It” stamina shots containing 250 mg nitrate per bottle (500 mg total) or two
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33 70 mL bottles with an isocaloric isonitrogenous black currant beverage and matched macronutrient
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composition. The first bottle was consumed 12 h before testing and the second bottle 2.5 h before
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38 the testing session. Participants performed 50 maximal knee extensions followed by passive knee
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40 extensions, divided into ten stages consisting of five consecutive contractions each. During the 50
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42 contractions across 35 subjects, there was no significant difference between the SUP and PLA
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44 conditions in mean peak torque and maximal work, indicating that SUP had no positive effects on
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47 muscle force generation and muscular work.
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49 3.3.2 Chronic Supplementation with Nitrate
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52 Acute Exercise Protocol
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55 Jonvik et al. (12) tested a 6-day SUP period in 15 recreationally-active male participants. Subjects
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57 ingested two 70 mL bottles of nitrate-rich (985 mg nitrate) or nitrate depleted (.37 mg nitrate)
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59 beetroot juice per day over breakfast and dinner. On day six (test day), participants ingested the last
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4 two SUP bottles exactly 3 h prior to the start of the performance tests. Participants were asked to
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6 perform three maximal voluntary isometric knee extensions, for 4 s, with 60 s rest intervals between
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9 successive attempts. Maximal knee extensor strength did not differ between the SUP and PLA
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11 groups during the isometric and isokinetic maximal power testing (12). Additionally, Kramer et al.
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13 (16) recruited 12 male CrossFit athletes administered SUP over 6-day periods consisting of either
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15 two potassium nitrate capsules each containing 260 mg nitrate (520 mg total per day), or two
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18 nitrate-free potassium chloride capsules separated into morning and evening doses. The final doses
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20 were consumed ≥24 h prior to performance testing, which assessed isokinetic quadriceps extension
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22 and hamstring flexion torque in two sets of five repetitions with 1-min rest between each set.
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24 Isometric contractions consisted of two sets of five repetitions with 5 s of rest between repetitions
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and 1 min rest between sets. The CrossFit benchmark workout, Grace, was also used, which
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29 consists of 30 clean-and-jerk repetitions against 61.37 kg as fast as possible. Maximal knee extensor
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31 strength and the Grace performance test revealed no differences between the SUP and PLA groups
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33 (16). Hoon et al. (10) recruited 19 physically active men (n=13) and women (n=6) volunteers for a
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study designed to examine the effects of nitrate SUP on muscle function (MF) and MVC. The SUP
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38 protocol consisted of the intake over 4 days of one 250 mL bottle of nitrate rich or nitrate-depleted
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40 beetroot juice (525 mg or 25 mg nitrate per bottle respectively) and another two bottles on the
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42 testing day 2 h and 4 h prior to exercise. Subjects performed four attempts of knee extensions at
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44 100% effort, with the greatest force over 0.9 s recorded as the maximum; each attempt was
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47 separated by 30 s of rest. No significant differences were observed in MF and MVC in SUP and
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49 PLA (10). Porcelli et al. (27) recruited seven healthy males recreationally involved in sports to
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51 examine the impacts of increased nitrate consumption through diet manipulation on MVC and
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53 MVT. The high nitrate diet included raw spinach, cooked collard greens, banana, and pomegranate
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56 juice leading to a total nitrate intake of 525 mg per day while the control group subjects followed
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58 their normal diet calculated to supply 185 mg of nitrate per day. Both diets were isoenergetic in
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60 accordance with subjects’ habitual energy intake and matched for physical activity levels, and
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2
3
4 contained similar proportions of macronutrients (55% CHO, 15% PRO, 30% FAT) except for
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6 nitrate. Dietary recommendations were followed for six days, with exercise testing on days five and
7
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9 six. Subjects were instructed to perform isometric knee extensions consisting of three MVCs of 3-4
10
11 s each. After each contraction, subjects rested for 2 min. The highest force was multiplied by the
12
13 moment arm to calculate MVT. No significant differences in MVT were observed between the high
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15 nitrate and control diet groups (27). Mosher et al. (22) supplemented 12 recreationally active males
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17
18 for 6 consecutive days with either 70 mL Beet It Sport® juice containing 400 mg nitrate or 70 mL
19
20 blackcurrant placebo drink (negligible nitrate level). Subjects performed sets of bench press
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22 repetitions to failure lifting a load of 60% 1RM, with each set separated by a 2-min rest period. In
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24 the SUP group, there was a significant improvement in the total number of repetitions in all three
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26
27
sets compared with the PLA group that led to a significantly greater number of total repetitions
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29 completed and total weight lifted (22). Additionally, Sim et al. (30) utilized a 12-month food
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31 frequency questionnaire submitted by 1,420 women to determine the amount of nitrate in their diets.
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33 Three different intake groups were established of ≥89.0 mg nitrate/day, 64.2 to <89.0 mg
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nitrate/day, or <64.2 mg nitrate/day. The nitrate source was starchy/non-starchy vegetables and
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37
38 meat (84.5% and 15.5% respectively). Upper limb muscular strength was determined by measuring
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40 the amount of force (kg) the forearm flexors can produce using a dynamometer in a grip strength
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42 test. Three test trials were performed using the participants’ dominant hand, and the peak value was
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44 recorded. A higher total nitrate intake was found associated with stronger grip strength, and a higher
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47 dietary nitrate was associated with a 16% reduced likelihood of weak grip strength (30).
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49 Chronic Exercise Protocol
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52 Fulford et al. (7) supplemented eight healthy, physically active male participants with two 250 mL
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54 bottles of Beet It Sport® beetroot juice or nitrate-depleted (25 mg of nitrate) beetroot juice taken in
55
56
57 the morning and evening containing 630 mg nitrate each for 15 days. On testing days, the entire 500
58
59 mL was ingested 2.5 h prior to the exercise protocol. Single leg-knee extension ergometry was
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 1 12
2
3
4 performed within a 1.5-T superconducting magnet: 112.2 s at baseline (not performing exercise),
5
6 followed by 50 MVC, cued from an audible signal, each repetition lasting 6.6 s with 2.2 s rest
7
8
9 between repetitions followed by 352 s recovery upon completion. The subjects attended the
10
11 laboratory to perform the exercise 2.5 h, 5 days, and 15 days after the start of SUP. No significant
12
13 SUP effect was found relative to PLA for MVC (mean over the entire 6.6 s of contractions) (7).
14
15
16
17
18
19
20
21
4. DISCUSSION
22
23
24 The main findings of this systematic review were that nitrate SUP may have an ergogenic effect on
25
26 muscle strength given the appropriate dosage, format, timing, and type of exercise assessment.
27
28
29 Muscle hypertrophy was not as widely assessed in the studies included and therefore these data are
30
31 inconclusive. Out of the 12 studies reviewed here, the results of six indicated that when participants
32
33 were given nitrate SUP or increased their total dietary intake of nitrate, muscle strength and
34
35 muscular performance were improved (22,24,28–30,38). We could therefore state that 50% of the
36
37
38 SUP protocols used in the selected studies led to some improvement, in line with the purpose of this
39
40 review. Notwithstanding, the remaining six SUP interventions resulted in no differences in muscle
41
42 hypertrophy or strength in the tested subjects (7,10,12,16). In this 50% of studies, regardless of
43
44 whether a nitrate supplement was used or dietary nitrate intake increased, SUP was ineffective at
45
46
47
improving muscle strength, hypertrophy or performance. Below we analyze the variables that could
48
49 have led to the different outcomes (acute vs. chronic, dose, duration, format, type of exercise, and
50
51 exercise test) with the goal of identifying those associated with an ergogenic effect of SUP.
52
53
54 4.1 Comparison Statement
55
56
57
The effects of nitrate SUP were associated with few comparator factors. Overall, the research
58
59 shared characteristics such as a small sample size, and similar age group, gender, health status, and
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 1 13
2
3
4 past injuries.
5
6
7 All but one investigation (30) included fewer than 40 participants. The age group weighted average
8
9 was 24.59 ± 4.29 (mean ± SD) years, which makes it generally a young population, with the
10
11 exception of one outlier study whose participants were aged 75.2 ± 2.4 (mean ± SD) years (30).
12
13
14 Physiological adaptations to resistance training may be enhanced in younger subjects, as recently
15
16 they have been found to show a greater increase in muscle mass in response to resistance training,
17
18 and age and muscle growth show negative correlation (25). Hence, the age group chosen for the
19
20 studies was appropriate to minimize misrepresentation of the results. Most of the studies had male
21
22
23 participants, although there was one outlier study including 1,420 women (30), and only three of the
24
25 12 studies included female subjects (10,18,30). The recruitment of female participants rather than
26
27 male for resistance training is on average much lower. However, when compared to young men,
28
29 older men and older women, young females exhibited greater muscle quality (MQ) improvements
30
31
32
following nine weeks of strength training (5). Only one of the three studies including female
33
34 participants detected a beneficial effect of increased nitrate ingestion on muscular strength (30). The
35
36 training status of participants of the individual studies varied widely. Those recreationally
37
38 physically active (22,28), involved in a specific combat sport (24), or resistance trained (29,38)
39
40
experienced muscle strength gains while the participants of another study (30) also showed
41
42
43 performance improvements although their level of physical activity was unclear. Conversely, the
44
45 studies detecting no effects of SUP also recruited recreationally active participants (7,10,12,18,27)
46
47 or sport specific CrossFit athletes (16). Thus, the training status of participants fails to explain the
48
49 discrepancy between those who exhibited strength improvements as a result to increased nitrate
50
51
52 intake and those who did not.
53
54 4.2 Supplementation Protocols
55
56
57 The different studies reviewed varied widely in their methods. In all studies, the trial design was
58
59 randomized, double-blind, controlled crossover, with the exception of two studies which featured a
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 1 14
2
3
4 randomized crossover (27) and randomized controlled (30) design. Supplement source and how it
5
6 was administered varied among studies. One study examined the impact of increasing total nitrate
7
8
9 ingested through a diet that included foods with a high nitrate content like beetroot, spinach, cooked
10
11 collard greens, banana, and pomegranate juice (27). Another study simply estimated total nitrate
12
13 ingestion of participants through a 12-month diet recall questionnaire (FFQ) (30). For the controls,
14
15 five studies provided a list of high nitrate foods to avoid either 24 hours (24,29) or 48 hours (18,28)
16
17
18 prior to exercise tests, or for the entirety of the study (16). Three studies (24,28,29) reported a
19
20 positive effect of nitrate SUP on muscle hypertrophy and strength. Further forms of SUP were
21
22 capsules (16), a beetroot-based gel (24), or nitrate-rich beetroot juice (7,10,12,18,22,28,29,38). In
23
24 six studies (16,18,24,27–29), participants were instructed to refrain from using antibacterial
25
26
27
mouthwash and chewing gum which could contain antimicrobials and negatively impact the
28
29 production of nitric oxide (8). Additionally, there appeared to be an optimal time-frame that seemed
30
31 most efficient for the SUP to have an effect. Of the six studies that observed an effect of nitrate
32
33 SUP, four of them utilized an acute SUP protocol (24,28,29,38) and doses of 800 mg, 756 mg, 400
34
35
mg, and 400 mg, respectively. In two, SUP was chronic and doses were 400 mg (22) and ≥ 89 mg
36
37
38 (30) per day. Mosher et al. (22) tested exercise performance on the last (sixth) day of SUP, allowing
39
40 for possible acute performance benefits. In contrast, of the six studies that observed no effects of
41
42 SUP only in one were participants subjected to acute SUP (18) at a dose of 500 mg per day.
43
44 Participants of the remaining five received chronic nitrate SUP (7,10,12,16) with daily doses of 985
45
46
47 mg, 520 mg, 525 mg, and 1260 mg, respectively and of 525 mg through dietary sources (27). With
48
49 the exception of one study (30), experimental designs were crossover and washout periods varied:
50
51 72 h (18,22,27,29,38), one week (10,12), 10 days (16), and 14 days (7). Porcelli et al. (27)
52
53 manipulated dietary nitrate ingestion and implemented a longer washout period of 20 days. In the
54
55
56 study by De Oliveira et al. (24) the washout period used was unclear. Bondonno et al. (2) conducted
57
58 a study to observe how long systemic nitrate and nitrite remain elevated following cessation of high
59
60 nitrate intake. Their results indicated that after 7 days of high nitrate intake, systemic nitrate and
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 1 15
2
3
4 nitrite levels fall after 2 days and return to baseline levels 7 days after cessation of nitrate intake
5
6 suggesting that daily nitrate ingestion is required to maintain high systemic nitrate and nitrite levels.
7
8
9 However, for the purpose of this review, these results also suggest that a 72 h washout out period is
10
11 sufficient.
12
13
14 4.3 Exercise Protocol
15
16 With regards to the training protocols used to test participants in the different studies, methods
17
18
19 varied in terms of number of sessions, time between them, as well as the type of exercise. All
20
21 studies involved a single exercise session with the exception of one involving multiple sessions.
22
23 Fulford et al. (7) utilized a single leg knee extension to measure MVC. Exercise was performed
24
25 within a 1.5-T superconducting magnet 2.5 h, 5 days, and 15 days after SUP. The dose of 630 mg
26
27
28 twice a day (1260 mg per day) was also the highest among the 12 studies reviewed with no effect
29
30 found at any testing time point. Those with a single exercise session made up the remaining 11
31
32 studies reviewed here, and used either a lower body or upper body resistance training protocol, or
33
34 both. Ranchal-Sanchez et al. (28) used an exercise protocol that incorporated both lower and upper
35
36
37
body resistance training through back squat and bench press. The back squat was performed at 60%,
38
39 70%, and 80% of the participants’ 1RM, assessing maximum repetitions completed at each
40
41 percentage maximal strength value, with 3 min of rest between efforts. In effect, there is evidence to
42
43 suggest that when strength training between 50%-90% of 1RM, rest intervals should be between 3
44
45
min and 5 min (6). There was an observed beneficial effect of nitrate SUP at 60% and 70% 1RM,
46
47
48 but not at 80% 1RM (28). In addition, there was no observed effect for the bench press exercise at
49
50 any percentage of maximal strength performed after 3 min of rest from back squats (28). Rodríguez-
51
52 Fernández et al. (29) had their participants complete a lower body-focused resistance training
53
54 exercise protocol testing maximal repetitions of half squats with different inertia loads, and also
55
56
57 incorporated a 3-min rest interval between efforts. A positive effect of nitrate SUP was detected on
58
59 total power output at all loads (29). In contrast, six studies (7,10,12,16,18,27) utilized single leg
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 1 16
2
3
4 extension as the exercise protocol and observed no impacts of nitrate SUP on muscle hypertrophy
5
6 or strength. This could be because their outcome measures were MF and MVC. Thus, while SUP is
7
8
9 believed to enhance muscle efficiency for a given amount of force production, it does not seem to
10
11 increase force levels as confirmed by Fulford et al. (7) by measuring phosphocreatine (PCr) cost
12
13 through p-magnetic resonance spectroscopy (P MRS) (7). The remaining four studies utilized an
14
15 upper body exercise protocol and, in all, a positive effect of SUP was observed. In the studies by
16
17
18 Sim et al. and De Oliveira et al. (24,30), the exercise protocol was a hand grip test and both groups
19
20 reported improved repeated grip strength in association with a higher nitrate intake. Williams et al.
21
22 (38) used the bench press as the resistance training exercise protocol, and had participants perform
23
24 repetitions at 70% of their 1RM at maximal power output, in addition to completing as many
25
26
27
repetitions as possible at 70% 1RM with 2-min of rest between efforts. A positive effect of SUP
28
29 was observed in both scenarios (38). Mosher et al. (22) used a similar exercise protocol in which
30
31 subjects had to complete sets of bench press repetitions until failure at 60% 1RM separated by 2-
32
33 min rest intervals. More effective muscle contractions with a lower overall energy cost were
34
35
reported, meaning that SUP helped conserve energy over the sets, thus allowing participants to
36
37
38 complete more sets. These data suggest that SUP could diminish muscular fatigue and improve
39
40 recovery.
41
42
43 4.4 Variables besides Muscle Hypertrophy and Strength
44
45
Most of the studies reviewed measured different dependent variables aside from muscle
46
47
48 hypertrophy and strength in their participants to find out whether nitrate SUP or increasing nitrate
49
50 intake could improve other performance markers such as: maximal aerobic capacity, muscular
51
52 endurance, PCr cost, velocity of lifts, and timed-up-and-go (TUG). This latter test measures the
53
54 time it takes an individual to rise from a chair of seat height 46 cm, walk 3 meters, and turn around
55
56
57 to return to sit on the chair. Fulford et al. (7) found a significant reduction in PCr cost per unit force
58
59 output for SUP at the end of exercise protocol as measured by a P MRS, thus confirming that SUP
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 1 17
2
3
4 reduces the PCr cost of force production and enhances muscle efficiency (7). Kramer et al. (16)
5
6 noted improved peak power output in a 30-s Wingate test, but no other beneficial effects of nitrate
7
8
9 SUP were observed. Sim et al. (30) found TUG performance to be marginally faster alongside
10
11 improved grip strength measurements as a result of a chronic high-nitrate diet. It should be noted
12
13 that the overall both calorie and protein intake were higher in the group of participants put on a high
14
15 nitrate diet, so this could have biased their results (30). Porcelli et al. (27) reported a significant
16
17
18 improvement manifesting as reduced VO2 values in the SUP group compared to PLA, but no effects
19
20 on MVC. Additionally, in some studies, plasma nitrate concentrations, phosphocreatine recovery
21
22 kinetics, blood lactate, O2 saturation, and RPE were measured. The only other outstanding
23
24 difference between the SUP and PLA groups in all the studies reviewed was an elevated plasma
25
26
27
nitrate concentration in the SUP groups of three of the studies (7,12,27).
28
29 4.5 Limitations
30
31
32 Overall, the studies selected for review had little to no bias and were therefore methodologically
33
34 sound. In half of the studies analyzed, a meaningful association emerged between nitrate SUP and
35
36
37
improved muscular strength, although some key aspects could have influenced the results. Ten of
38
39 the twelve included studies included a relatively small sample size (N<20). Even though all but one
40
41 of the studies was of a cross-over design, they included relatively few participants. Ten studies
42
43 implemented a double-blind design, except for two which were unclear (27,30). In a similar
44
45
manner, the population of participants chosen varied in their level of physical activity. Further,
46
47
48 variations in sources and doses of nitrate SUP also precluded a uniform pattern of investigation thus
49
50 providing contradictory results.
51
52
53 Another potential limitation of the studies was that dietary nitrate was not always taken into
54
55 account. In six studies, participant food intake was determined before the experiment or for the
56
57 entirety of the study. While no differences were detected in the consumption of nitrates in
58
59 participants, biased responses in the food recall questionnaires is another possibility. Another factor
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62
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 1 18
2
3
4 that was not consistently accounted for in the studies were sleep patterns. Evidence showed that
5
6 poor sleep or sleep deprivation negatively impacts muscle strength and performance (15). Further,
7
8
9 menstrual cycle stage was not reported and this could be a limitation when trying to determine if
10
11 SUP improved muscle hypertrophy and strength in female participants (10,18,30). There is
12
13 evidence that resistance training during the first two weeks of the menstrual cycle is more beneficial
14
15 to optimize training than the last two weeks (37). Moreover, the relative lack of hypertrophy
16
17
18 observed within the studies may be due to the inconsistencies of male and female participants, as
19
20 females typically show less muscle hypertrophy than males when undergoing resistance exercise
21
22 interventions.
23
24
25 Having examined in detail all relevant characteristics of the different studies, we can compare the
26
27 findings of our research to those of other meta-analyses and systematic reviews with similar
28
29 objectives. A review of supplements with purported effects on muscle mass and strength concluded
30
31
32
that SUP in general had an acute beneficial effect on muscle strength and increased muscle
33
34 efficiency for a given amount of force produced, measured as PCr cost per unit force output (7),
35
36 although it was not found to increase force levels (7,35). In addition, SUP reduced energy cost and
37
38 improved time to exhaustion during low and high intensity muscle contractions (35). Another
39
40
review observed the potential benefits of beetroot juice on intermittent high-intensity exercise
41
42
43 efforts such as resistance training or repeated sprints (4). The authors of this review concluded that
44
45 SUP could diminish muscular fatigue associated with high-intensity exercise efforts but that the
46
47 mechanism behind this was still unclear (4). They also argued that the potentially improved release
48
49 and reuptake of calcium at the sarcoplasmic reticulum could help power production associated with
50
51
52 improvements in muscle shortening velocity (4). However, this hypothesized mechanism was based
53
54 on the results of a study conducted in rats and therefore requires confirmation in humans (36).
55
56 Overall, their conclusions are in line with the findings of the present review related to the study
57
58 designs and protocols used in those reporting a positive effect of SUP. More research is needed to
59
60
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 1 19
2
3
4 test the impacts of a more standardized SUP source, dose, timing, and exercise protocol including
5
6 mechanistic studies assessing changes in muscle function after acute nitrate ingestion.
7
8
9 The main findings of this systematic review point to an ergogenic effect of nitrate SUP under
10
11 certain conditions of dosage, format, timing, and type of exercise test implemented. Impacts on
12
13
14 exercise involving low and high intensity muscle contractions may consist of improved muscle
15
16 efficiency, as reflected by sparing of PCr and reducing energy expenditure per unit of force output
17
18 confirmed by P MRS (7), and a shorter time to exhaustion. In 67% of the studies that detected a
19
20 beneficial effect of nitrate SUP, the exercise test implemented involved compound movements
21
22
23 (back squat and/or bench press) at 60%-70% of 1RM, and SUP was both acute and chronic and
24
25 consisted of at least 400 mg nitrate spread across x days or per day. To improve data consistency,
26
27 this loading percentage and dose need to be further explored. In addition, the beneficial impacts of
28
29 this SUP were always related to muscular strength, so potential benefits on muscle hypertrophy
30
31
32
have yet to be determined.
33
34
35
36
37 5. PRACTICAL APPLICATIONS
38
39
40 We consider this systematic review is of particular interest for clinicians, practitioners, and
41
42 consumers who seek to better understand the ergogenic benefits for resistance training of
43
44 supplementation with nitrate. Thus, an ergogenic effect of nitrate was produced in response to an
45
46
47
acute dose of 400 mg nitrate, in the form of beetroot juice/shot, administered 2 to 2.5 h before
48
49 exercise. When the resistance training test involved compound movements conducted at 60-70%
50
51 1RM, muscle efficiency and time to exhaustion were improved when performing sets of repetitions
52
53 to failure through sparing of PCr and energy cost reduction.
54
55
56 Further studies are needed to examine the more chronic effects of acute nitrate SUP at the dose and
57
58 timing detailed above. This will help establish whether the initial ergogenic effect observed in a
59
60
single exercise session continues over a chronic training program and thus provides training
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 1 20
2
3
4 benefits and/or adaptations in the long term. Large longitudinal studies will also help identify a
5
6 possible hypertrophic effect.
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3
4 REFERENCES
5
6
7 1. Anderson, JE. A role for nitric oxide in muscle repair: nitric oxide-mediated activation of
8
9 muscle satellite cells. Mol Biol Cell 11: 1859–1874, 2000.
10
11
12 2. Bondonno, CP, Liu, AH, Croft, KD, Ward, NC, Puddey, IB, Woodman, RJ, et al. Short-term
13
14 effects of a high nitrate diet on nitrate metabolism in healthy individuals. Nutrients 7: 1906–
15
16 1915, 2015.
17
18
19 3. Cermak, NM, Gibala, MJ, and van Loon, LJC. Nitrate supplementation’s improvement of
20
21 10-km time-trial performance in trained cyclists. Int J Sport Nutr Exerc Metab 22: 64–71,
22
23
24 2012.
25
26 4. Domínguez, R, Maté-Muñoz, JL, Cuenca, E, García-Fernández, P, Mata-Ordoñez, F,
27
28
29 Lozano-Estevan, MC, et al. Effects of beetroot juice supplementation on intermittent high-
30
31 intensity exercise efforts. J Int Soc Sports Nutr 15: 2, 2018.
32
33
34 5. Dutta, C. Commentary on “Effects of strength training and detraining on muscle quality:
35
36 Age and gender comparisons.” Journals Gerontol - Ser A Biol Sci Med Sci 55: 152–157,
37
38 2000.
39
40
41 6. Freitas de Salles, B, Simão, R, Miranda, F, da Silva Novaes, J, Lemos, A, and Willardson,
42
43 JM. Rest Interval between Sets in Strength Training. Sport Med 39: 765–777,
44
45 2009.Available from: https://doi.org/10.2165/11315230-000000000-00000
46
47
48 7. Fulford, J, Winyard, PG, Vanhatalo, A, Bailey, SJ, Blackwell, JR, and Jones, AM. Influence
49
50 of dietary nitrate supplementation on human skeletal muscle metabolism and force
51
52
53 production during maximum voluntary contractions. Pflugers Arch 465: 517–528, 2013.
54
55 8. Govoni, M, Jansson, EA, Weitzberg, E, and Lundberg, JO. The increase in plasma nitrite
56
57
58 after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric
59
60 oxide Biol Chem 19: 333–337, 2008.
61
62
63
64
65
 1 22
2
3
4 9. Higgins, JPT, Altman, DG, Gøtzsche, PC, Jüni, P, Moher, D, Oxman, AD, et al. The
5
6 Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. BMJ 343: 1–9,
7
8
9 2011.
10
11 10. Hoon, MW, Fornusek, C, Chapman, PG, and Johnson, NA. The effect of nitrate
12
13
14 supplementation on muscle contraction in healthy adults. Eur J Sport Sci 15: 712–719, 2015.
15
16 11. Hys, K. Identification of the Reasons Why Individual Consumers Purchase Dietary
17
18
19 Supplements. In: Perspectives on Consumer Behaviour: Theoretical Aspects and Practical
20
21 Applications. Sroka, W, ed. . Cham: Springer International Publishing, 2020. pp. 193–
22
23 209Available from: https://doi.org/10.1007/978-3-030-47380-8_9
24
25
26 12. Jonvik, KL, Hoogervorst, D, Peelen, HB, de Niet, M, Verdijk, LB, van Loon, LJC, et al. The
27
28 impact of beetroot juice supplementation on muscular endurance, maximal strength and
29
30 countermovement jump performance. Eur J Sport Sci 1–8, 2020.
31
32
33 13. Kelly, J, Vanhatalo, A, Wilkerson, DP, Wylie, LJ, and Jones, AM. Effects of nitrate on the
34
35 power-duration relationship for severe-intensity exercise. Med Sci Sports Exerc 45: 1798–
36
37
38 1806, 2013.
39
40 14. Kerksick, CM, Wilborn, CD, Roberts, MD, Smith-Ryan, A, Kleiner, SM, Jäger, R, et al.
41
42
43 ISSN exercise & sports nutrition review update: Research & recommendations. J Int Soc
44
45 Sports Nutr 15: 1–57, 2018.
46
47
48 15. Knowles, OE, Drinkwater, EJ, Urwin, CS, Lamon, S, and Aisbett, B. Inadequate sleep and
49
50 muscle strength: Implications for resistance training. J Sci Med Sport 21: 959–968,
51
52 2018.Available from: https://doi.org/10.1016/j.jsams.2018.01.012
53
54
55 16. Kramer, SJ, Baur, DA, Spicer, MT, Vukovich, MD, and Ormsbee, MJ. The effect of six days
56
57 of dietary nitrate supplementation on performance in trained CrossFit athletes. J Int Soc
58
59 Sports Nutr 13: 39, 2016.
60
61
62
63
64
65
 1 23
2
3
4 17. Lansley, KE, Winyard, PG, Bailey, SJ, Vanhatalo, A, Wilkerson, DP, Blackwell, JR, et al.
5
6 Acute dietary nitrate supplementation improves cycling time trial performance. Med Sci
7
8
9 Sports Exerc 43: 1125–1131, 2011.
10
11 18. Lee, S, Abel, MG, Thomas, T, Symons, TB, and Yates, JW. Acute beetroot juice
12
13
14 supplementation does not attenuate knee extensor exercise muscle fatigue in a healthy young
15
16 population. J Exerc Nutr Biochem 23: 55–62, 2019.
17
18
19 19. Lorenzo Calvo, J, Alorda-Capo, F, Pareja-Galeano, H, Jiménez, SL, Calvo, JL, Alorda-
20
21 Capo, F, et al. Influence of Nitrate Supplementation on Endurance Cyclic Sports
22
23 Performance: A Systematic Review. Nutrients 12, 2020.Available from: %3CGo
24
25
26 20. Moher, D, Liberati, A, Tetzlaff, J, and Altman, DG. Preferred reporting items for systematic
27
28 reviews and meta-analyses: the PRISMA statement. PLoS Med 6: e1000097, 2009.
29
30
31 21. Moncada, S and Higgs, A. The L-arginine-nitric oxide pathway. N Engl J Med 329: 2002–
32
33 2012, 1993.
34
35
36 22. Mosher, SL, Sparks, SA, Williams, EL, Bentley, DJ, and Mc Naughton, LR. Ingestion of a
37
38 Nitric Oxide Enhancing Supplement Improves Resistance Exercise Performance. J strength
39
40 Cond Res 30: 3520–3524, 2016.
41
42
43 23. de Oliveira, GV, do Nascimento, LAD, Volino-Souza, M, do Couto Vellozo, O, Alvares,
44
45 TS, Vieira de Oliveira, G, et al. A single oral dose of beetroot-based gel does not improve
46
47
48 muscle oxygenation parameters, but speeds up handgrip isometric strength recovery in
49
50 recreational combat sports athletes. Biol Sport 37: 93–99, 2020.Available from:
51
52 https://search.ebscohost.com/login.aspx?direct=true&db=sph&AN=141869690&site=ehost-
53
54 live&scope=site
55
56
57 24. de Oliveira, GV, do Nascimento, LAD, Volino-Souza, M, do Couto Vellozo, O, Alvares,
58
59 TS, Vieira de Oliveira, G, et al. A single oral dose of beetroot-based gel does not improve
60
61
62
63
64
65
 1 24
2
3
4 muscle oxygenation parameters, but speeds up handgrip isometric strength recovery in
5
6 recreational combat sports athletes. Biol Sport 37: 93–99, 2020.
7
8
9 25. Phillips, BE, Williams, JP, Greenhaff, PL, Smith, K, and Atherton, PJ. Physiological
10
11 adaptations to resistance exercise as a function of age. JCI insight 2: 1–16, 2017.
12
13
14 26. Pinna, M, Roberto, S, Milia, R, Marongiu, E, Olla, S, Loi, A, et al. Effect of beetroot juice
15
16 supplementation on aerobic response during swimming. Nutrients 6: 605–615, 2014.
17
18
19 27. Porcelli, S, Pugliese, L, Rejc, E, Pavei, G, Bonato, M, Montorsi, M, et al. Effects of a Short-
20
21 Term High-Nitrate Diet on Exercise Performance. Nutrients 8, 2016.
22
23
24 28. Ranchal-Sanchez, A, Diaz-Bernier, VM, De La Florida-Villagran, CA, Llorente-Cantarero,
25
26 FJ, Campos-Perez, J, and Jurado-Castro, JM. Acute Effects of Beetroot Juice Supplements
27
28
29 on Resistance Training: A Randomized Double-Blind Crossover. Nutrients 12, 2020.
30
31 29. Rodríguez-Fernández, A, Castillo, D, Raya-González, J, Domínguez, R, and Bailey, SJ.
32
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34 Beetroot juice supplementation increases concentric and eccentric muscle power output.
35
36 Original investigation. J Sci Med Sport , 2020.
37
38
39 30. Sim, M, Lewis, JR, Blekkenhorst, LC, Bondonno, CP, Devine, A, Zhu, K, et al. Dietary
40
41 nitrate intake is associated with muscle function in older women. J Cachexia Sarcopenia
42
43 Muscle 10: 601–610, 2019.
44
45
46 31. Smith, LW, Smith, JD, and Criswell, DS. Involvement of nitric oxide synthase in skeletal
47
48 muscle adaptation to chronic overload. J Appl Physiol 92: 2005–2011, 2002.
49
50
51 32. Stamler, JS and Meissner, G. Physiology of nitric oxide in skeletal muscle. Physiol Rev 81:
52
53 209–237, 2001.
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56 33. Trewin, CB, Hopkins, WG, and Pyne, DB. Relationship between world-ranking and
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58 Olympic performance of swimmers. J Sports Sci 22: 339–345, 2004.Available from:
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9 35. Valenzuela, PL, Morales, JS, Emanuele, E, Pareja-Galeano, H, and Lucia, A. Supplements
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11 with purported effects on muscle mass and strength. Eur J Nutr 58: 2983–3008,
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14 2019.Available from: http://dx.doi.org/10.1007/s00394-018-1882-z
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16 36. Whitfield, J, Ludzki, A, Heigenhauser, GJF, Senden, JMG, Verdijk, LB, van Loon, LJC, et
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19 al. Beetroot juice supplementation reduces whole body oxygen consumption but does not
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21 improve indices of mitochondrial efficiency in human skeletal muscle. J Physiol 594: 421–
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23 435, 2016.
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26 37. Wikström-Frisén, L, Boraxbekk, CJ, and Henriksson-Larsén, K. Effects on power, strength
27
28 and lean body mass of menstrual/oral contraceptive cycle based resistance training. J Sports
29
30 Med Phys Fitness 57: 43–52, 2017.
31
32
33 38. Williams, TD, Martin, MP, Mintz, JA, Rogers, RR, and Ballmann, CG. Effect of Acute
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35 Beetroot Juice Supplementation on Bench Press Power, Velocity, and Repetition Volume. J
36
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Table 1

Table 1 Risk of bias graph

Blinding of Blindness of Incomplete Selective

Sequences Allocation Other
participants and outcome outcome outcome
generation concealment sources
Study personnel assessment data reporting
(selection (selection of
(performance (detection (attrition (reporting
bias) bias) bias
bias) bias) bias) bias)

De Oliveira et al., 2020 (23) + ? + ? + + ?

Fulford et al., 2013 (7) + ? + ? + + ?

Hoon et al., 2015 (10) + + + ? + + ?

Jonvik et al., 2020 (12) + + + ? + + ?

Kramer et al., 2016 (16) + ? + + + + ?

Lee et al., 2019 (18) + + + ? + + ?

Mosher et al., 2016 (22) + + + ? + + ?

Porcelli et al., 2016 (26) + ? ? ? + + ?

Ranchal-Sanchez et al., 2020 (27) + + + ? + + ?

Rodríguez-Ferdández et al., 2020 (28) + + + ? + + ?

 2

Sim et al., 2019 (29) + ? ? ? + + ?

Williams et al., 2020 (37) + + + ? + + ?

[+] Low risk of bias [-] High risk of bias [?] Unclear risk of bias
Table 2

Table 2 Summary of the studies included in the systematic review examining the effects of nitrates on muscle hypertrophy and strength

Study Subjects Supplementation and protocols Exercise protocol Results

De Oliveira et 14 physically active  SUP: Beetroot gel. 3 sets of handgrip (40% of MVC). Forearm MVC: SUP
al., 2020 (23) men.  PLA: Nitrate-depleted gel. > PLA
Crossover design. Acute: 756mg nitrate 120min before exercise.

Lee et al., 2019 35 physically active  SUP: Beet It stamina shot. - Maximal knee extensions (10x5 MKES: SUP = PLA
(18) subjects (9 women).  PLA: Nitrate depleted black currant drink. reps).
Parallel design. Acute: 250mg nitrate 12h and 2.5h before exercise.
Ranchal- 12 resistance trained  SUP: BJ. - Back squat (3 sets: 60%, 70%, and RTF back squat (60%
Sanchez et al., men.  PLA: Nitrate depleted black currant drink. 80% 1RM). and 70% 1RM): SUP
2020 (27) Crossover design. Acute: 400mg nitrate 2h before exercise. - Bench press (3 sets: 60%, 70%, and > PLA
80% 1RM). RTF back squat (80%
1RM): SUP = PLA
RTF bench press:
SUP = PLA
Rodríguez- 18 physically active  SUP: Beetroot shots. - Half squat (4x8 reps with a different Power output: SUP >
Fernández et al., men.  PLA: Nitrate depleted drink (<10mg). moment inertia). PLA
2020 (28) Crossover design. Acute: 800mg nitrate 2.5h before exercise.
Williams et al., 11 resistance trained  SUP: BJ. - Bench press (RTF at 70% 1RM). Power output: SUP >
2020 (37) men. Crossover  PLA: Blackcurrant drink (negligible nitrate). PLA
design. Acute: 800mg nitrate 2h before exercise. RTF: SUP > PLA

Kramer et al., 12 male CrossFit  SUP: Potassium nitrate capsules. Day 1: MKES: SUP = PLA
2016 (16) athletes.  PLA: Nitrate-free potassium chloride capsules. - Quadriceps extension and hamstring CrossFit
 2

Crossover design. Chronic: 520mg/day potassium nitrate (twice daily) for flexion (2×5 reps). performance: SUP =
6 days. Last dose ≥24h before exercise test. - Isometric contractions (2×5 reps). PLA
Day 2:
- Grace performance test: clean-and-
jerk (30 reps with 61.37kg as fast as
possible).
Mosher et al., 12 physically active  SUP: Beet It Sport shot. - Bench press (60% 1RM). RTF: SUP > PLA
2016 (22) men.  PLA: Blackcurrant drink (negligible nitrate). Total weight lifted:
Crossover design. Chronic: 400mg/day nitrate for 6 days. SUP > PLA

Porcelli et al., 7 physically active  SUP: Nitrate-rich diet. - Knee extension (3 reps at MVC). MVC: SUP = PLA
2016 (26) men.  PLA: Normal diet (~185mg). MVT: SUP = PLA
Crossover design. Chronic: 525mg/day nitrate for 6 days.
Sim et al., 2019 1420 women:  SUP1: ≥89.0mg/day nitrate through nitrate-rich diet. - Grip strength. Grip strength: SUP >
(29) SUP1 (N=473)  SUP2: 64.2 to <89.0mg/day nitrate through nitrate- CON
SUP2 (N=474) medium diet.
CON: (N=473)  CON: <64.2mg/day nitrate through nitrate-poor diet.
Cross-sectional Chronic: Nitrate intake through diet for 12 month.
design.
Fulford et al., 8 physically active  SUP: BJ. - Single leg knee extension performed MVC: SUP = PLA
2013 (7) men.  PLA: Nitrate depleted BJ (25mg). 2.5h, 5 days, and 15 days after MKES: SUP = PLA
Crossover design. Chronic: 1260mg/day nitrate (twice daily) for 15 days. supplementation.
Acute: 1260mg nitrate 2.5h before exercise.
Hoon et al., 19 physically active  SUP: Nitrate Max - Knee extension (4 reps) MF: SUP = PLA
2015 (10) subjects (6 women).  PLA: Nitrate depleted Nitrate Max (25mg). MVC: SUP = PLA
Crossover design. Chronic: 525mg/day nitrate for 4 days.
 3

Acute: 525mg nitrate 4h and 2h before exercise.

Jonvik et al., 15 physically active  SUP: BJ. - Isometric knee extensions (3 reps for MKES: SUP = PLA
2020 (12) men.  PLA: Nitrate depleted BJ (0.37mg). 4s at MVC).
Crossover design. Chronic: 1970mg/day nitrate (twice daily) for 6 days.
Acute: 1970mg nitrate 3h before exercise.
BJ= beetroot juice, CON= Control group, MF= muscle function, MKES= maximal knee extensor strength, MSR= muscle strength recovery, MVC=

maximal voluntary contraction, MVT= maximal voluntary torque, PLA= placebo group, RM= repetition maximum, RTF = repetitions to failure, SUP=

supplementation group.
Figure 1

Records identified through

Identification

database searching (n = 862)

PubMed = 236
WOS = 378 Records identified through
MEDLINE Complete = 248 manual searching (n = 5)

Records screened after duplicates removed (n = 457)

Studies excluded by title and abstract (n =

Screening

205):
-Reviews/meta-analysis/book/letter (n = 50)
-Studies in animals (n = 67)
-Pathology (n = 54)
-Other supplements (n = 34)
-Non full text available (n = 8)

Full-text articles assessed for eligibility (n = 39)

Eligibility

Full-text articles excluded (n = 27)

-Did not measure body composition or muscle
function (n = 24)
-Non-blind or randomized (n = 0)
-Unknown dose of supplementation (n = 0)
-Use of nitrate with another supplement (n = 3)
-Did not use nitrate supplement (n = 0)
Included

-Studies without physical activity (n = 0)

Articles included in systematic review (n = 12)

Figure 2