Higher endogenous nitrite levels are associated with superior exercise capacity in highly trained athletes

Nitric Oxide 27 (2012) 75–81

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Higher endogenous nitrite levels are associated with superior exercise capacity in highly trained athletes Matthias Totzeck a, Ulrike B. Hendgen-Cotta a, Christos Rammos a, Lisa-Marie Frommke a, Christian Knackstedt b, Hans-Georg Predel c, Malte Kelm a, Tienush Rassaf a,⇑ a b c

Department of Medicine, Division of Cardiology, Pulmonary Diseases and Vascular Medicine, Medical Faculty, University Hospital Düsseldorf, Düsseldorf, Germany Department of Cardiology, University Hospital Maastricht, Maastricht, The Netherlands Institute of Cardiology and Sport Medicine, German Sport University, Cologne, Germany

a r t i c l e

i n f o

Article history: Received 16 March 2012 Revised 6 May 2012 Available online 18 May 2012 Keywords: Nitrite Exercise capacity Endothelial function

a b s t r a c t Factors improving exercise capacity in highly trained individuals are of major interest. Recent studies suggest that the dietary intake of inorganic nitrate may enhance athletic performance. This has been related to the stepwise in vivo bioactivation of nitrate to nitrite and nitric oxide (NO) with the modulation of mitochondrial function. Here we show that higher baseline levels of nitrite are associated with a superior exercise capacity in highly trained athletes independent of endothelial function. Eleven male athletes were enrolled in this investigation and each participant reported twice to the testing facility (total of n = 22 observations). Venous blood was obtained to determine the levels of circulating plasma nitrite and nitrate. Endothelial function was assessed by measuring flow-mediated vasodilation (FMD). Hereafter, participants completed a stepwise bicycle exercise test until exhaustion. Blood was drawn from the ear lope to determine the levels of lactate. Lactate anaerobic thresholds (LAT) in relation to heart rate were calculated using non-linear regression models. Baseline plasma nitrite levels correlated with LATs (r = 0.65; p = 0.001, n = 22) and with endothelial function as assessed by FMD (r = 0.71; p = 0.0002). Correlation coefficients from both testing days did not differ. Multiple linear regressions showed that baseline plasma nitrite level but not endothelial function was an independent predictor of exercise capacity. No such correlations were determined for plasma nitrate levels. Ó 2012 Elsevier Inc. All rights reserved.

Introduction Exercise capacity is determined by a coordinated interplay between the cardiovascular, respiratory and nervous system, blood and muscles [1]. There are still controversies regarding possible factors that limit exercise performance and whether these factors can be measured and modulated. One central theory supports the notion that the overall ability of an individual to remain in an aerobic state during exercise reflects exercise capacity [2]. Increasing workloads require the muscles to produce adequately higher amounts of energy substrates, namely ATP. However, with more intense exercise, myocytes will not be able to maintain this process using aerobic respiration. Quite consequently, the exercising muscle will utilize anaerobic glycolysis, which produces ATP more Abbreviations: NO, nitric oxide; eNOS, endothelial NO synthase; FMD, flowmediated dilation; LAT, lactate anaerobic threshold. ⇑ Corresponding author. Address: University Hospital Düsseldorf, Medical Faculty, Division of Cardiology, Pulmonology and Vascular Medicine, Moorenstrasse 5, D-40225 Düsseldorf, Germany. Fax: +49 211 811 8912. E-mail address: [email protected] (T. Rassaf). 1089-8603/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.niox.2012.05.003

rapidly at the cost of increased lactate generation. Once the bodily capacities of lactate elimination are exhausted, lactate is accumulated in the circulation. This in turn has a negative and limiting impact on muscle function and has been termed anaerobic threshold. Endurance exercise training aims to increase the capability of an athlete to maintain aerobic metabolism, and changes in lactate anaerobic threshold are one of the most important measures to evaluate the effectiveness of training routines [2]. However, mechanisms that increase the ability to exercise below anaerobic thresholds are incompletely understood. Nitric oxide (NO) is a gaseous signaling molecule that contributes to the regulation of a wide variety of processes in the cardiovascular, nervous and immune system [3–5]. NO is enzymatically generated through three NO synthase isoforms. Particularly for the maintenance of cardiovascular function endothelial NO synthase (eNOS) activity is essential. Nitrite as the main oxidation product in human plasma sensitively reflects eNOS-dependent NO production under certain conditions [6]. We recently demonstrated that impaired eNOS function, either through its pharmacological inhibition [7] or in patients with endothelial dysfunction

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[8,9], significantly impairs NO bioavailability, deteriorates vascular function and finally leads to a much-reduced exercise capacity. Under physiological hypoxia, e.g. in the exercising muscle, generation of NO by NOS may be impaired due to a lack of oxygen as essential cofactor. Whilst previously regarded to be an inert product of NO metabolism, nitrite is now suggested to provide an alternative source of bioactive NO. Along the physiological oxygen gradient, nitrite may be reduced back to NO involving a variety of different mechanism [10–16]. Apart from NOS dependent nitrite generation, bodily nitrite levels also derive from nutritional sources. After the intake of inorganic nitrate, as highly concentrated in green leafy vegetables, oral cavity resident bacteria reduce inorganic nitrate to nitrite, which is continuously swallowed and absorbed [17,18]. Exogenous supplementation of nitrate has recently been demonstrated to improve endurance exercise capacity mainly by decreasing the amount of oxygen cost [19–26]. These studies demonstrated that intake of nitrate increased circulating nitrite levels and reduced the uptake of oxygen needed for delivering comparable amounts of work. This has been attributed to an optimization of mitochondrial respiration with increased rates of ATP formed in relation to otherwise constant oxygen consumption (P/O gradients) [19]. The reduction of dietary nitrate is therefore regarded to be an alternative pathway for NO generation that may modulate the ability to exercise in aerobic states. While dietary nitrate increased plasma nitrite levels approximately 2–4-fold leading to the documented benefits for exercise capacity, the role of endogenous nitrite levels on exercise capacity is not known. It therefore remains elusive whether baseline endogenous nitrite levels correlate with exercise capacity as measured by anaerobic thresholds, and whether this may provide advantages to those individuals with higher levels. Although the mentioned studies suggest that nitrate treatment optimizes skeletal muscle metabolism and respiration, it has also been demonstrated that elevation of circulating nitrite affects other components relevant for exercise, e.g. endothelial function. Nitrite levels correlate with endothelial function, which can be measured by means of ultrasound-guided assessment of endothelium dependent maximum dilation using FMD technique [27,28]. Improved endothelial and thus vascular function and the ability to circulate oxygen to the exercising muscle may therefore be another contributor to enhanced athlete’s performance with higher nitrite levels. We here show that baseline plasma nitrite levels correlate with exercise capacity and vascular function in a selected collective of young, healthy male athletes. Using multivariate linear regression models we furthermore demonstrate that baseline nitrite predicts exercise capacity independent of an improvement of vascular function.

Table 1 Subjects characteristics and blood parameters. Means ± S.D. Age (a) Height (cm) Weight (kg) Body mass index Training units/week Watt maximum (W/kg)

24 ± 2 185 + 5 79 ± 5 23.1 ± 1.2 5±1 4.2 ± 0.6

Chemistry Panel Sodium (mmol/l) Calcium (mmol/l) Chloride (mmol/l) Creatinine (mg/dl) Uric acid mg/dl) Total cholesterol (mg) Triglycerides (mg/dl) Total protein (g/dl) C-reactive protein (mg/dl) HbA1c (%)

139 ± 3 2.3 ± 0.1 103 ± 1 1.0 ± 0.1 5.5 ± 0.6 174 ± 29 117 ± 79 7.1 ± 0.2 <0.03 5.3 ± 0.2

Blood Count Leucocytes (104/ll) Red blood cells (x106/ll) Hemoglobin (g/dl) Hematocrit (%) Platelets (/ll)

5.1 ± 1.8 5.2 ± 0.3 15.4 ± 0.9 45 ± 3 217 ± 44

were either competitive cyclists or triathletes and thus familiar with the testing devices. Baseline participants’ characteristics including training details are shown in Table 1. All subjects were asked to remain on their regular exercise routine and daily diet. Nitrate supplements were not allowed during the entire study. Subjects were asked to fasten 12 h before both testing days. All tests were conducted between 7.00 and 11.00 am. Upon arrival at the testing laboratory, subjects received a standardized breakfast on both testing days consisting of 500 ml mineral water and one cheese bun. The participants were hereafter asked to rest in a supine position before a blood draw from the cubital vein was conducted. 15 min later FMD was assessed on the contralateral arm using ultrasound technique [7,8,29,30]. This was followed by measuring blood pressure. Upon completion of this protocol step, subjects were challenged with a step-wise ergometric testing on a stationary bicycle ergometer [31]. In order to corroborate the results of day 1 and to obtain an independent correlation on two consecutive occasions, 7 days later each participant repeated the procedures providing a total of n = 22 observations. All participants gave written consent to the study procedures as well as to the data handling prior to the first testing. The responsible ethics committee at the Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany, had approved all procedures.

Materials and methods Blood parameters Study subjects 11 male athletes were enrolled in this study applying the specific in- and exclusion criteria as listed in Fig. 1. All participants

Blood samples were taken from the left anticubital vein for the determination of clinical parameters and for the assessment of the circulating NO pool (nitrite and nitrate).

Fig. 1. In- and exclusion criteria. Subjects (n = 11) were selected based on their training status and whether they were accustomed to the experimental set up including stationary bicycle ergometers. Accordingly, only cyclists and triathletes were included if all further criteria were met.

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Clinical parameters: Using tubes from clinical routine (BD Vacutainer), heparin-plasma and EDTA-full blood aliquots were obtained for blood cell counts and full chemistry panels. All further analyses were performed by the Institute of Clinical Chemistry and Laboratory Diagnostic, University Hospital Duesseldorf. Plasma nitrite and nitrate: The procedures for the determination of plasma nitrite and nitrate levels were identical to those described previously [32–34]. Briefly, heparinized full blood was centrifuged at 800 g for 10 min (4° Celcius). The resulting plasma aliquots were snap frozen in liquid nitrogen and stored at 80 °Celsius until further analysis. All samples were incubated at a 1:1 ratio with methanol to precipitate plasma proteins. Supernatants were analyzed by HPLC (ENO-20) technique according to the manufacturer’s guidelines and calibrated with standard nitrite/nitrate solutions (Sigma–Aldrich, Taufkirchen, Germany). FMD FMD of the right brachial artery was determined using a standardized ultrasound-based protocol (12-MHz transducer, GE vivid i) followed by an automated analysis [27]. A forearm blood-pressure cuff was placed around the forearm and inflated to 200 mm Hg for 5 min. Diameter and Doppler-flow velocity were determined at baseline and after cuff deflation at 20, 40, 60 and 80 s. Using an automated system to measure diameters (Brachial Analyzer; Medical Imaging Applications, Iowa City, Iowa, USA) low variability was seen in our methodology [7]. FMD values were expressed as the maximum percentage change from baseline. Exercise tests All tests were performed in an air-conditioned room (22–23 °C) on a stationary bicycle ergometer (Excalibur Sport V 2006, Lode B.V., the Netherlands). The ergometer was attached to a laptop computer for a continuous acquisition of cycling parameters (Lode Ergometry Manager). Subjects used their own cycling shoe-pedal clip system during all exercise tests. Specific ergometric set-ups were recorded and applied on both testing occasions. If requested, cooling through a ventilator was allowed. All participants were asked to complete an ergometric test on both testing days. Following a warm-up period (90 W for 5 min) and a resting period (5 min), the ergometric test was started at 100 W and increased by 50 W every 5 min [31]. The ergometer was set to a hyperbolic mode delivering the same amount of Watt independent of cycled rounds per min (rpm). However, the participants were asked to maintain their optimal rpm during all exercise tests. The heart rate (beats per min, bpm) was continuously measured by a wireless transmitter around the thorax of the participants (Polar Systems). At the end of each step, subjects rated physical exhaustion according to the Borg RPE scale and a blood sample (20 ll) was obtained from the ear lobe to determine lactate concentrations. The protocol was continued until physical exhaustion. Lactate anaerobic thresholds (4 mM) were calculated using non-linear regression models (Winlactat software, Mesics, Germany). Statistics

Fisher Z-transform, and the z value was used to calculate the level of significance. Multilinear regression was conducted with FMD and nitrite as independent (forced entry) and LAT as dependent variable to determine if FMD and nitrite may serve as independent predictors of exercise capacity. All statistical tests were conducted using SPSS 20.0 and Graph Pad Prism 5.0 for Mac OS. Results Baseline characteristics of study group Table 1 lists salient anthropometric measures of the subjects included in this study. Systolic blood pressure averaged 129 ± 8 mm Hg and diastolic blood pressure 81 ± 5 mm Hg. No participant was on a regular medications and no chronic or acute disease was noted. Average maximum power achieved in the stepwise ergometric test was 328 ± 60 W equaling 4.2 ± 0.6 W per kg body weight. Each participant was tested on two occasions providing a total of n = 22 observations. For these n = 22 observations (day one and two), lactate anaerobic thresholds (4 mM) in relation to heart rate averaged 163 ± 16 bpm, FMD values exhibited a mean of 7.3 ± 0.5% maximum dilation in relation to the resting diameter and nitrite levels averaged 0.092 ± 0.008 lM while nitrate levels were 50 ± 6 lM in all subjects. Notably, a tendency to higher nitrate levels were detected on day two which did not reach significance. Respective separate values for both testing days and mean values for all n = 22 observations are displayed in Table 2. Correlation of nitrite, nitrate and vascular function with exercise capacity Univariate analysis (Table 3) showed that baseline nitrite levels of the subjects correlate with exercise capacity as assessed by lactate anaerobic threshold (0.65, p = 0.001; Fig. 2A). To further assess whether the correlation was the same on two consecutive occasions, correlation coefficients for day 1 and day 2 were analyzed and showed no statistical difference (day 1 vs day 2: r = 0. 68 vs r = 0.58; p = 0.719, n = 11; Fig. 2B and C). In addition, no significant differences in LAT (Fig. 2D) and endogenous plasma nitrite levels (2E) were detected comparing the results from day 1 and 2. Finally,

Table 2 Values (means ± S.D.) for nitrite, nitrate, FMD and LAT on both testing days (11 participants tested twice) and averaged total values.

Nitrite (lM) Nitrate (lM) FMD (%) LAT (bpm)

Day 1

Day 2

Average

p day 1 vs day 2

0.084 ± 0.042 40 ± 18 6.4 ± 2 160 ± 17

0.101 ± 0.036 61 ± 37 8.2 ± 1 166 ± 15

0.092 ± 0.008 50 ± 6 7.3 ± 0.5 163 ± 16

0.362 0.085 0.002 0.316

FMD, flow mediated dilation; LAT, lactate anaerobic threshold.

Table 3 Univariate analysis. LAT

Data are presented as means ± S.D. All data were checked for normal distribution and no departures were noted. Differences between day 1 and 2 were compared using Student’s two-tailed t-test for paired observations. Correlation between individual parameters were calculated using univariate analyses. Results are expressed as Pearson’s r and corresponding p values. In order to compare the association between nitrite and LAT (day 1 vs. day 2), the two correlation coefficients were transformed with the

Nitrite (lM) Nitrate (lM) FMD (%) Hb (g/dl) BMI (kg/m2)

FMD

r

p

r

p

0.651 0.292 0.458 0.039 0.164

0.001 0.187 0.032 0.863 0.465

0.714 0.447

<0.0001 0.037

0.125 0.032

0.578 0.889

FMD, flow mediated dilation; LAT, lactate anaerobic threshold, Hb, hemoglobin; BMI, body mass index.

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b Lactate anaerobic threshold (bpm)

a 220

200

1 2 3 4 5 6 7 8 9 10 11

180

160

140

120

180 100

160

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Nitrite (µM)

c

140

Lactate anaerobic threshold (bpm)

Lactate anaerobic thershold (bpm)

200

120

100 0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Nitrite [µM]

200

1 2 3 4 5 6 7 8 9 10 11

180

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100 0.04

0.06

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Nitrite (µM)

d

e

LAT

Nitrite 0.4

220 200

0.3

µM

bpm

180 160

0.2

140

0.1 120 100

0.0 day 1

day 2

day 1

day 2

Fig. 2. Correlation of baseline plasma nitrite and exercise capacity as measured by lactate anaerobic threshold (4 mM) at the corresponding heart rate in beats per minute (bpm). (A) Values for all n = 22 observations. (B) Values for day one and day two (C) with no significant differences in correlation coefficients. Symbols represent all 11 participants. (D) Lactate anaerobic thresholds (LAT) and endogenous plasma nitrite levels (E) showed no significant difference comparing day one and two (p = 0.31 and p = 0.36, respectively.

no correlation in regard to exercise was detected between nitrate and LAT (r = 0.29, p = 0.19). We subsequently assessed vascular function in each participant using ultrasound based FMD technique. Nitrite levels in these athletes correlated with the FMD at rest values (r = 0.71, p = 0.0002; Fig. 3) which is in line with previous observations [35]. Concentrations of nitrate correlated with FMD although at lower significance levels (r = 0.447, p = 0.037), an observation previously not described. Finally, vascular function also correlated with exercise capacity (Pearson r = 0.46, p = 0.0323, Fig. 4). Univariate analysis

between nitrite and the variables nitrate, height, weight, body mass index (BMI), hemoglobin (Hb), age, and blood pressure revealed no further significant correlations. Nitrite is a predictor of exercise capacity independent of changes in vascular function Both Nitrite and FMD correlated with exercise capacity. In order to determine whether both parameters are predictors of exercise capacity, multivariate regression analysis was performed. Table 4

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shows the calculated coefficients for nitrite and FMD. While nitrite is an independent predictor, no such result was determined for vascular function as assessed by FMD. This suggests that baseline nitrite levels predict exercise capacity most likely by affecting other mechanisms than vascular function relevant for a maximum athletic performance. We have additionally performed a multivariate analysis with FMD as dependent and nitrite and nitrate as independent variables. This showed that nitrite but not nitrate is an independent predictor of FMD (Table 4).

Discussion The key findings of this study are (a) baseline endogenous nitrite levels correlate positively with exercise capacity as measured by anaerobic thresholds and with vascular function as determined by FMD in highly trained athletes, (b) baseline nitrite is an independent predictor of exercise capacity and (c) this relation may be independent of nitrite-related improvements of vascular functions.

Fig. 3. Correlation of baseline plasma nitrite and vascular function as assessed by ultrasound guided flow mediated dilation (Pearson r = 0.71, p = 0.0002).

Fig. 4. Correlation of vascular function as assessed by ultrasound guided flow mediated dilation and exercise capacity as measured by lactate anaerobic threshold (4 mM) at the corresponding heart rate in beats per minute (bpm) (Pearson r = 0.46, p = 0.0323).

Table 4 Multivariate linear regression analysis. LAT

Nitrite (lM) Nitrate (lM) FMD (%) Adjusted r2 ANOVA

FMD

Standard coefficient

p

Standard coefficient

p

0.661 – 0.014 0.363 –

0.016 – 0.954 – 0.005

0.637 0.250 – 0.52 –

0.001 0.133 – – <0.0001

FMD, flow mediated dilation; LAT, lactate anaerobic threshold.

Nitrite and exercise capacity A coordinated interplay between several components of the cardiovascular system is indispensible for an adequate regulation during physical exercise. One central controversy remaining is whether markers exist that might indicate limitations in individuals although undergoing optimal training routines. The contribution of NO in the signaling of numerous cardiovascular functions has been well documented [3]. Whilst previously considered waste products of NO metabolism, nitrate and particularly nitrite are now widely accepted as major sources of NO apart from NOS related NO production [36]. Endogenous nitrite levels derive from two sources: Two thirds of plasma nitrite levels relate to endogenous NO generation from NOS and the remainder to exogenous dietary supplementation mainly through nitrate [37,38]. We have previously shown that nitrite levels may reflect NOS activity in fastened individuals. However, it remains unclear whether baseline nitrite levels are also tightly correlated with functional aspects of the cardiovascular system. Quite surprisingly it has been demonstrated by several independent groups that the intake of dietary nitrate has profound and sustained effects on cardiovascular functions in humans. This relates to baseline cardiovascular functions such as reduced blood pressures in otherwise healthy, non-hypertensive subjects [17] as well as to athletic performance in exercise physiology studies [19–26,39]. The latter body of studies comprises short-term exercise protocols, endurance testing such as time trials as well as muscle contractile efficiency investigations – all of which were substantially influenced under the intake of dietary nitrate. While all of these trials examined the effects between baseline and supplemented nitrite levels, which were increased 2–4-fold in many of the corresponding investigations, it is not known whether baseline nitrite levels correlate with athletic performance. We here showed that endogenous pre-exercise nitrite levels are associated with superior exercise capacity as measured by lactate anaerobic thresholds. Confirming the general notion that nitrite is highly significant for the regulation of cardiovascular function, these results extend the existing knowledge to baseline nitrite levels. Critically, in a previous investigation, we were not able to establish a comparable relation [7]. However, this former study included both trained and untrained individuals as well as patients with proven endothelial dysfunction including impaired NO generation. By contrast, this concurrent study’s selection criteria were restricted to

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highly trained, healthy, male individuals with low variation in health status, anthropometric measures and exercise capacity.

capacity. Finally, we cannot rule out any influence on cardiac functions, which must be assessed in future studies using invasive or non-invasive cardiac imaging techniques.

Nitrite predicts exercise capacity independent of vascular functions The nitrate-nitrite-NO pathway is emerging as an alternative route for the generation of NO [37]. While nitrate is reduced by oral bacterial flora to nitrite, the resulting nitrite may be reduced along oxygen and physiological pH gradients thus contributing substantially to NO bioavailability. Intriguingly, nitrite bioactivation is therefore limited to events of physiological hypoxia, e.g. during exercise, and to pathophysiological events, such as tissue ischemia in myocardial infarction. Several mechanisms that activate nitrite to NO have been identified including Hb in red blood cells and myoglobin in muscles [12,13]. The dependence on NO signaling by dietary nitrate/nitrite supplementation in humans has been evidenced by both the activation of the canonical NO/soluable guanylate cyclase/cyclic guanosine monophosphate pathway [40] and by cGMP-independent regulation of mitochondrial efficiency. The latter novel findings were determined using human biopsies from athletes receiving nitrate supplementation and exhibiting improved performance [19]. Exercise is accompanied by a physiological decrease in oxygen levels. This may lead to activation of nitrite to NO as well as other reactive nitrogen intermediates. These products have been suggested to regulate mitochondrial efficiency, e.g. by the downregulation of adenine nucleotide translocase and by reaction with complexes of the respiratory change. The authors concluded that these signal transduction mechanisms regulated the generation of mitochondria derived reactive oxygen species and lead to an optimized ATP generation using less amounts of oxygen, a mechanism previously not seen with any other substance available in diets. This study showed a distinct regulation of muscle function when administrating nitrate to healthy young participants. It also implied that the exercise-improving effects from nitrate may be largely detectable within the musculature itself. In line with this study, our present investigation demonstrates that nitrite is an independent predictor of exercise capacity. Using multivariate linear regression models, we show that this relation does not rely on an improvement of conduit artery function as measured by FMD. Conclusively, nitrite may be an additional marker for exercise capacity in highly trained individuals signaling superior performance in those with elevated baseline values. These investigations further contribute to the existing knowledge that the nitrate-nitrite-NO pathway may be a target for a dietary modulation of cardiovascular functions. Study limitations Our present study has included both measures for exercise capacity and vascular function. In the scope of exercise capacity, lactate anaerobic thresholds may provide robust results for overall athletic performance. However, further studies using more endurance related field-testings are required to further assess the relation between the circulating NO pool in plasma and naturally also in whole blood and exercise capacity in different endurance and maximum exercise disciplines. In addition, we assessed vascular function in conduit arteries of athletes. Between the two study days, significant differences were detected using statistical tests for paired observations. Notably, reliability of FMD technique was good (r = 0.862, p = 0.001) in this present study, but further investigations will be needed to determine vascular functions in this cohort particularly with regard to training routines and competitions. Further studies are also needed to investigate whether nitrite may influence the microcirculation, which undergoes much higher oxygen/pH decreases during exercise and thus may impact exercise

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