Influence of Nitrate Supplementation on Endurance Cyclic Sports Performance: A Systematic Review

urance can be de ned as the capacity to maintain one's velocity or power output for the longest possible time. Maintaining such activity can lead to the onset of fatigue. Dietary nitrate supplementation produces an ergogenic e ect due to the improvement of mitochondrial oxygen e ciency through a reduction in the oxygen cost of exercise that increases vasodilation and blood ow to the skeletal muscle in recreationally active subjects. However, the e ects of dietary nitrate supplementation on well-trained endurance athletes remain unclear; such supplementation could a ect more performance areas. In the present study, a systematic review of the literature was conducted to clarify the use and e ects of nitrate as a dietary supplement in endurance athletes trained in cyclic sports (repetitive movement sports). A systematic search was carried out following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines in the databases of SCOPUS, Web of Science (WOS), Medline (PubMed), and Sport Discus from 1 January 2010 to 30 November 2019. Twenty-seven studies were included in the study. The methodological quality of the articles was assessed using the McMaster Critical Review Form. Statistically signi cant ergogenic results were obtained in 8 (29.63%) of the 27 studies investigated, with signi cant results obtained for cardiorespiratory parameters and performance measures. Improvement in exercise tolerance was obtained, which could help with exhaustion over time, while the improvement in exercise economics was not as clear. Additionally, the dose necessary for this ergogenic e ect seems to have a direct relationship with the physical condition of the athlete. The

27 studies investigated, with signi cant results obtained for cardiorespiratory parameters and performance measures. Improvement in exercise tolerance was obtained, which could help with exhaustion over time, while the improvement in exercise economics was not as clear. Additionally, the dose necessary for this ergogenic e ect seems to have a direct relationship with the physical condition of the athlete. The acute dose is around 6–12.4 mmol/day of nitrate administered 2–3 h before the activity, with the same amount given as a chronic dose over 6–15 days. Further studies are required to understand the factors that a ect the potential ergogenic impacts of nitrate on athletic performance among endurance athletes. Keywords:nitrate; nitric oxide; beetroot juice; performance exercise; endurance; cyclic 1. Introduction Nitrate supplementation enhances nitric oxide (NO) bioavailability via the NO3–nitrite–NO pathway, which is involved in several physiological processes that could potentially improve skeletal muscle function [1,2]. NO endogenous synthesis occurs through at least two di erent physiological pathways: the NO synthase (NOS)-dependent and NOS-independent pathways [2]. L-arginine and L-citrulline are the main precursors of NO in the NOS-dependent pathway, while nitrate (NO3 ) and nitrite (NO2 ) are the main substrates that produce NO via the NOS-independent pathway (Figure). Nutrients2020,12, 1796; doi:10.3390 /nu12061796 /journal/nutrients

Nutrients2020,12, 1796 2 of 20Nutrients 2020, 12, x FOR PEER REVIEW 2 of 23 Figure 1. The main beneficial effects of nitric oxide on physical performance. In addition to the endogenous generation of NO through the NOS pathway, NO3 − and NO2 − reserves can also be increased exogenically through one’s diet. Approximately 80% of the nitrates in one’s diet come from the consumption of vegetables [3], mainly through green-leaf vegetables, such as lettuce, spinach, arugula, celery, watercress, and beet. Beet is one of the main nitrate sources since it contains 250 mg (4 mmol) of nitrate per 100 g of fresh weight (Table 1). Table 1. Classification of vegetables according to nitrate content *. Nitrate Content (mg/100 g Fresh Weight) Vegetable Varieties Very low, <20 Artichoke, asparagus, broad bean, eggplant, garlic, onion, green bean, mushroom, pea, pepper, potato, summer squash, sweet potato, tomato, watermelon Low, 20 to <50 Broccoli, carrot, cauliflower, cucumber, pumpkin, chicory Middle, 50 to <100 Cabbage, dill, turnip, savoy cabbage High, 100 to <250 Celeriac, Chinese cabbage, endive, fennel, kohlrabi, leek, parsley Very high, >250 Celery, cress, chervil, lettuce, red beetroot, spinach, rocket * Information obtained from Hord et al. 2009 [4]. NO signaling affects multiple physiological mechanisms that could potentially enhance skeletal muscle. NO mediates smooth muscle relaxation, which promotes vasodilation and blood flow regulation and thereby improves oxygen delivery and mitochondrial respiration (Figure 1). This, in turn, enhances type II muscle fiber function [5–7]. This physiological facilitation suggests that supplementation with nitrates could have ergogenic effects on cardiorespiratory endurance [8]. The ergogenic effects of nitrate have been demonstrated in endurance and submaximal exercises to improve high-intensity endurance performance [8,9]. This has led the scientific community to increase the number of investigations that analyze the ergogenic potential of nitrates under different exercise conditions, including high to low intensity, long to short duration, continuous and intermittent, normoxia and hypoxia, and acute dose and chronic exposure, with different nitrate sources and doses [10–13]. Supplementation with nitrates, either with pure nitrate salts or foods rich in nitrates, has been proven to enhance the sporting performance of recreationally active

ergogenic potential of nitrates under different exercise conditions, including high to low intensity, long to short duration, continuous and intermittent, normoxia and hypoxia, and acute dose and chronic exposure, with different nitrate sources and doses [10–13]. Supplementation with nitrates, either with pure nitrate salts or foods rich in nitrates, has been proven to enhance the sporting performance of recreationally active young people [14–17], while nitrate supplementation has not yet been shown to improve performance among non-professional athletes [18]. Figure 1.The main bene cial e ects of nitric oxide on physical performance. In addition to the endogenous generation of NO through the NOS pathway, NO3 and NO2 reserves can also be increased exogenically through one's diet. Approximately 80% of the nitrates in one's diet come from the consumption of vegetables [3], mainly through green-leaf vegetables, such as lettuce, spinach, arugula, celery, watercress, and beet. Beet is one of the main nitrate sources since it contains 250 mg (4 mmol) of nitrate per 100 g of fresh weight (Table). Table 1.Classi cation of vegetables according to nitrate content *. Nitrate Content (mg/100 g Fresh Weight) Vegetable Varieties Very low,<20 Artichoke, asparagus, broad bean, eggplant, garlic, onion, green bean, mushroom, pea, pepper, potato, summer squash, sweet potato, tomato, watermelon Low, 20 to<50 Broccoli, carrot, cauli ower, cucumber, pumpkin, chicory Middle, 50 to<100 Cabbage, dill, turnip, savoy cabbage High, 100 to<250 Celeriac, Chinese cabbage, endive, fennel, kohlrabi, leek, parsley Very high,>250 Celery, cress, chervil, lettuce, red beetroot, spinach, rocket * Information obtained from Hord et al. 2009 [4]. NO signaling a ects multiple physiological mechanisms that could potentially enhance skeletal muscle. NO mediates smooth muscle relaxation, which promotes vasodilation and blood ow regulation and thereby improves oxygen delivery and mitochondrial respiration (Figure). This, in turn, enhances type II muscle ber function [5–7]. This physiological facilitation suggests that supplementation with nitrates could have ergogenic e ects on cardiorespiratory endurance [8]. The ergogenic e ects of nitrate have been demonstrated in endurance and submaximal exercises to improve high-intensity endurance performance [8,9]. This has led the scienti c community to increase the number of investigations that analyze

turn, enhances type II muscle ber function [5–7]. This physiological facilitation suggests that supplementation with nitrates could have ergogenic e ects on cardiorespiratory endurance [8]. The ergogenic e ects of nitrate have been demonstrated in endurance and submaximal exercises to improve high-intensity endurance performance [8,9]. This has led the scienti c community to increase the number of investigations that analyze the ergogenic potential of nitrates under di erent exercise conditions, including high to low intensity, long to short duration, continuous and intermittent, normoxia and hypoxia, and acute dose and chronic exposure, with di erent nitrate sources and doses [10–13]. Supplementation with nitrates, either with pure nitrate salts or foods rich in nitrates, has been proven to enhance the sporting performance of recreationally active young people [14–17], while nitrate supplementation has not yet been shown to improve performance among non-professional athletes [18]. Endurance cyclic sports require repetitive movements, usually lasting more than ve minutes, through predominantly aerobic metabolism. When an endurance athlete takes part in high-intensity e ort, various physiological factors limit their performance, such as the maximum oxygen uptake,

Nutrients2020,12, 1796 3 of 20 rst and second ventilatory threshold, and energy e ciency and economy [8]. At the same time, these factors can potentially be improved through NO signaling via nitrate supplementation. The objective of this systematic review was to analyze the ergogenic e ect of nitrate supplementation on well-trained cyclic sports athletes, as well as to study the optimal sources and doses of nitrates. 2. Materials and Methods 2.1. Eligibility Criteria This systematic review focused on the e ect of nitrate supplementation on endurance cyclic sports performance. This review followed the PICOS question model for framing the research question and de ning the inclusion criteria [19]: P (population): “endurance and healthy trained athletes between 18 and 50 years old”; I (intervention): “supplementation with nitrate”; C (comparison): “same conditions with placebo or control group”; O (outcomes): “performance measures”; S (study design): “double- or single-blind design and randomized parallel or crossed.” 2.2. Literature Search A structured search was carried out following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines [20] in the SCOPUS, Web of Science (WOS), Sport Discus, and Medline (PubMed) databases. The search included original articles with randomized controlled crossover or parallel designs in which the intake of nitrate administered before and/or after the exercise was compared with an identical placebo situation. Due to the increasing number of investigations over the last 10 years interested in using nitrate as a supplement in athletes, the search was bounded from 1 January 2010 to 30 November 2019. Search terms were established matching the Medical Subject Headings (MeSH), as follows: “nitrate,” “nitric oxide,” “beetroot juice,” “performance exercise,” and “endurance.” The connectors OR and AND were also used to lter the search. 2.3. Study Selection The following inclusion criteria were applied to select studies: (I) articles depicting a well-designed experiment that included the ingestion of a dose of nitrate, or nitric oxide or beetroot juice before and/or during exercise in healthy trained athletes; (II) the study investigated endurance cyclic sports; (III) there was an identical experimental situation with or without the ingestion of a placebo in a normoxic condition;

were applied to select studies: (I) articles depicting a well-designed experiment that included the ingestion of a dose of nitrate, or nitric oxide or beetroot juice before and/or during exercise in healthy trained athletes; (II) the study investigated endurance cyclic sports; (III) there was an identical experimental situation with or without the ingestion of a placebo in a normoxic condition; (IV) there was a double- or single-blind design and randomized parallel or crossed design; (V) crossed over studies that did not allow for a clean-up period of over 24 h were excluded; (VI) there was clear information on the administration of nitrate; (VII) nitrate was administered in the form of a beverage, gum, or pills; (VIII) at least one of the measured performance variables was changes (e ort shown, maximum power, average power, time, distance, oxygen consumption, saturated oxygen, total work, or number of completed sprints among others); and (IX) the language was restricted to English. The following exclusion criteria were applied: (I) animal studies, (II) uncontrolled trials, (III) Zstudies using non-standardized turmeric extracts or extracts of unknown nitrate content, (IV) studies performed on non-trained or injured/sick subjects, (V) studies investigating children and the elderly, and (VI) studies in non-speci c sports or non-endurance sports. 2.4. Data Extraction The following information was extracted from the selected articles: study source (authors and year of publication), participant characteristics (level of activity or sports discipline, experience and training load described by the authors, number of participants and sex), age, VO2max, supplementation protocol (source of nitrates and duration), nitrate dose, time from the last dose of nitrate, exercise protocol, main outcome (performance measures), and results.

Nutrients2020,12, 1796 4 of 20 2.5. Quality Assessment and Risk of Bias The methodological quality of the articles, evaluated using McMaster's Critical Review Form [21], ranged between 13 and 15 points, representing a minimum methodological quality of 86.6% and a maximum of 100%. Of the 27 studies, 3 achieved 13 items, representing 11.11% of the total number of studies, 3 achieved 14 items, representing the same percentage of 11.11%, and the rest of the studies (21) achieved 15 items, representing 77.78% of the total number of studies. All studies achieved a “very good” quality. No study was excluded because it did not reach the minimum quality threshold. The main de ciencies found in methodological quality were associated with items 13 and 14 of the questionnaire and comprised the report of clinical importance and one study had drop-outs. The objective of this evaluation was to determine and to compare the quality between the di erent study designs (Table). Table 2.Methodological quality of the studies included in the systematic review. Reference 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ts % MQ Balsalobre et al. [22] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Bescâs et al. [18] 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 14 93.3VG Bescâs et al. [23] 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 14 93.3VG Boorsma et al. [24] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Callahan et al. [25] 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 13 86.6VG Cermak et al. [26] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Cermak et al. [27] 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 13 86.6VG Christense et al. [28] 1 1 1 1 1 1 1 1 1

0 1 13 86.6VG Cermak et al. [26] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Cermak et al. [27] 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 13 86.6VG Christense et al. [28] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Esen et al. [29] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Garnacho et al. [30] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Glaister et al. [31] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Hoon et al. [32] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Hoon et al. [33] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Lane et al. [34] 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 14 93.3VG Lansley et al. [35] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Lowings et al. [36] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG MacLeod et al. [37] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG McQuillan et al. [38] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG McQuillan et al. [39] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Nyakayiru et al. [40] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Nybäck et al. [41] 1 1 1 1 1 1 1 1 1 1 1 1 1 1

al. [39] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Nyakayiru et al. [40] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Nybäck et al. [41] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Pawlak et al. [13] 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 13 86.6VG Peacock et al. [42] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Pinna et al. [43] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Rokkedal et al. [44] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Shannon et al. [45] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Wilkerson et al. [46] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 100 VG Ts—Total items ful lled by study; 1—Criterion met; 0—Criterion not met; MQ—Methodological quality: P—poor 8 points, A—acceptable 9–10 points, G—good 11–12 points, VG—very good 13–15 points. 3. Results 3.1. Study Selection The initial research obtained through database searches provided 4341 records. Of these records, 3102 duplicates were removed. Of the 1239 remaining records, a total of 1067 were excluded after screening the titles and abstracts for eligibility. Ultimately, 27 studies were included in this review. The PRISMA ow chart was used to summarize the systematic research and selection process (Figure).

Nutrients2020,12, 1796 5 of 20Nutrients 2020, 12, x FOR PEER REVIEW 6 of 23 Keywords: nitrate, nitric oxide, beetroot juice, performance exercise, endurance Search limited to: 2010 - 2019 Humans Between 18 - 50 years PubMed (n = 1104) Medline Complete (n = 943) Scopus (n = 787) Web of Science (n = 882) SPORTDiscus (n = 625) TOTAL n = 4341 Duplicat records removed (n = 3102) Records screened (n = 1239) Studies removed by title/summary (n = 1067) Fully analyzed studies (n = 172) Studies included in the systematic review (n = 27) Excluded studies and reasons for exclusion (n = 140) Identification Screening Eligibility Included Figure 2. Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) ow. Summary of the systematic search and the study selection process.

Nutrients2020,12, 1796 6 of 20 3.2. Characteristics of the Studies A total of 323 participants were included in these 27 studies. The average sample included 12 subjects but seven studies assessed the e ects of nitrate in larger group subjects (up to 26 participants). The study mode was carried out using eight subjects. All subjects in the review were de ned by the authors as either trained or well-trained using the various criteria listed in Table. Among the 323 experimental subjects, only 39 (12.07%) were women. Moreover, female athletes only took part in 5 of the 27 studies selected, with only one study exclusively on trained women. Most of the studies were performed with populations in their 20s and 30s. Most focused on cycling, with 9 studies (33.33%) and 101 participants (31.27%), followed by combined cycling and triathlon studies, with 97 participants (30%) and 6 studies (22.2%). We also found other less represented sports, such as swimming (3), athletics (2), rowing (1), and cross-country skiing (2). For the ergogenic e ect of nitrate supplementation, statistically signi cant results were obtained in eight (29.63%) of the studies that investigated cyclic endurance sports performance [22,23,26,33,35,44,45]. The main form of supplementation was beetroot, except for three studies that used sodium nitrate [18,23,40] and one that used potassium nitrate [42]. Most of the studies used mmol as a measuring unit to determine the value of the dose of nitrate intake. The average dose of nitrate was 8.7 mmol/day (minimum 4 mmol/day and a 19.5 mmol/day maximum). Both acute and chronic supplementation protocols were found in the reviewed studies. Of the studies, 40.74% used chronic supplementation and 51.85% used acute supplementation, applying equally high and low doses for both acute and chronic treatments, and 7.4% used both types of supplementation. Studies on chronic treatment were performed over an average of 6 days, taking into account the fact that some treatments lasted 3 days, and others, such as those described by Balsalobre et al. [22], applied supplementation for 15 days. The last dose of nitrate before physical performance testing was not given at the same moment

A systematic review of nitrate supplementation effects on endurance performance in cyclic sports.