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Accepted Manuscript Physiological and performance effects of nitrate supplementation during roller-skiing in normoxia and normobaric hypoxia Linn Nybäck, Caroline Glännerud, Gustav Larsson, Eddie Weitzberg, Oliver Michael Shannon, Kerry McGawley PII:

S1089-8603(17)30088-5

DOI:

10.1016/j.niox.2017.08.001

Reference:

YNIOX 1690

To appear in:

Nitric Oxide

Received Date: 15 March 2017 Revised Date:

31 July 2017

Accepted Date: 2 August 2017

Please cite this article as: L. Nybäck, C. Glännerud, G. Larsson, E. Weitzberg, O.M. Shannon, K. McGawley, Physiological and performance effects of nitrate supplementation during roller-skiing in normoxia and normobaric hypoxia, Nitric Oxide (2017), doi: 10.1016/j.niox.2017.08.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Physiological and performance effects of nitrate supplementation during roller-skiing in

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normoxia and normobaric hypoxia

3 Linn Nybäcka, Caroline Glänneruda, Gustav Larssona, Eddie Weitzbergb, Oliver Michael

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Shannonc, Kerry McGawleya.

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University, 831 25 Östersund, Sweden

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Swedish Winter Sports Research Centre, Department of Health Sciences, Mid Sweden

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Sweden

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Leeds, LS163QS, United Kingdom

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Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm,

Research Institute for Sport, Physical Activity, and Leisure, Leeds Beckett University,

13 Corresponding author:

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Kerry McGawley, Swedish Winter Sports Research Centre, Department of Health Sciences,

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Mid Sweden University, 831 25 Östersund, Sweden

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Telephone: +46 70 399 98 74

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Fax: +46 10 142 80 04

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E-mail: [email protected]

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Running head: Nitrate supplementation and exercise in hypoxia

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ABSTRACT

23 The present study examined the effects of acute nitrate (NO3-) supplementation ingested in the

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form of concentrated beetroot juice on cross-country roller-ski performance in normoxia (N)

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and normobaric hypoxia (H). Eight competitive cross-country skiers (five males: age 22 ± 3

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years, V̇ O2max 71.5 ± 4.7 mL·kg-1·min-1; three females: age 21 ± 1 years, V̇ O2max 58.4 ± 2.5

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mL·kg-1·min-1) were supplemented with a single dose of NO3--rich beetroot juice (BRJ, ~ 13

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mmol NO3-) or a NO3--depleted placebo (PL, ~ 0 mmol NO3-) and performed 2 x 6-min

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submaximal exercise bouts and a 1000-m time-trial (TT) on a treadmill in N (20.9% O2) or H

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(16.8% O2). The four experimental trials were presented in a randomised, counter-balanced

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order. Plasma NO3- and nitrite concentrations were significantly higher following BRJ

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compared to PL (both p < 0.001). However, respiratory variables, heart rate, blood lactate

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concentration, ratings of perceived exertion, and near-infrared spectroscopy-derived measures

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of muscle tissue oxygenation during submaximal exercise were not significantly different

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between BRJ and PL (all p > 0.05). Likewise, time to complete the TT was unaffected by

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supplementation in both N and H (p > 0.05). In conclusion, an acute dose of ~ 13 mmol NO3-

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does not affect physiological or performance responses to submaximal or maximal treadmill

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roller-skiing in competitive cross-country skiers exercising in N and H.

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Key words: beetroot juice; endurance exercise; simulated altitude; work economy

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1. INTRODUCTION

43 Dietary nitrate (NO3-) supplementation, often administered in the form of beetroot juice or

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nitrate salts, has not only demonstrated health benefits such as lowering blood pressure (BP)

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[1], but has also been shown to improve exercise time to exhaustion (TTE) and time-trial (TT)

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performance [2–4]. In healthy adults, a single dose of 5.2 mmol NO3- ingested 2.5 h prior to

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exercise lowered resting BP and reduced the oxygen (O2) cost of moderate-intensity exercise

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[3], whereas continuous supplementation for six days with a similar dose also improved the

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oxygenation status of exercising skeletal muscle and tolerance to high-intensity exercise [5–

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7].

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In well-trained athletes the effects of NO3- supplementation are less clear. Cyclists produced a

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greater power output for the same O2 uptake (V̇ O2) and demonstrated improved 4- and 16.1-

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km TT performances after ingesting 6.2 mmol of NO3- 2.75 h prior to exercise [8].

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Conversely, elite cross-country skiers did not reduce the submaximal O2 cost or 5-km TT

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performance time during running exercise following an acute dose of 9.9 mmol NO3- [9].

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Moreover, aerobic fitness levels and responsiveness to NO3- supplementation (in terms of

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exercise performance) have recently been reported to be negatively correlated [10]. Despite

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this, a number studies have shown individual improvements following NO3- ingestion in well-

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trained athletes, identifying these individuals as so-called “responders” [11–13].

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The effects of NO3- supplementation are commonly explained by the reduction of NO3- to

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nitrite (NO2-), and subsequently nitric oxide (NO), a signalling molecule involved in multiple

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physiological functions [14,15]. NO is generally produced via endogenous utilization of L-

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arginine by the NO synthase (NOS) enzymes using O2 and multiple cofactors [16]. This

ACCEPTED MANUSCRIPT pathway is O2-dependent and therefore attenuated in hypoxic conditions [14], which may be

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relevant during exercise at altitude. In contrast to the L-arginine–NOS pathway, the NO3--

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NO2--NO pathway is O2-independent and is therefore believed to compensate for the altered

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endogenous NO production under hypoxic conditions [14]. A reduced O2 availability (e.g.,

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during exercise at altitude) also impairs exercise capacity. Thus, supplementation with NO3-

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prior to exercising in hypoxia may help to combat lowered NO production and the reduced

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capacity for exercise performance.

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Previous studies have reported multiple beneficial physiological and performance effects of

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NO3- supplementation in varying degrees of hypoxia, using simulated altitudes of 2500–5000

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m. These effects include a reduction in steady-state V̇ O2 [17–20], an increase in arterial O2

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saturation (SpO2) [17,20], an elevation of near-infrared spectroscopy- (NIRS-) derived muscle

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tissue oxygenation [17], and a reduction in muscle metabolic perturbations [21]. Likewise,

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NO3- supplementation has also been reported to enhance exercise TTE [17,19,21] and TT

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performance [18,20] in hypoxia. Importantly, individuals who showed no effects of NO3-

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supplementation in normoxia improved their tolerance to severe-intensity exercise in hypoxia

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[19], or maintained their performance capacity seen in normoxia also in hypoxia following

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NO3- ingestion [21]. Furthermore, NO3- supplementation has been shown to elicit comparable

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reductions in steady-state V̇ O2 and improvements in 1500-m running TT performance in

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individuals with a broad range of aerobic fitness levels exercising in moderate hypoxia (~

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2500 m simulated altitude [20]. It is therefore conceivable that participants not responding to

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NO3- supplementation in normoxic conditions (such as trained athletes) might still benefit

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from NO3- ingestion in hypoxia.

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ACCEPTED MANUSCRIPT Most research regarding endurance exercise performance and NO3- supplementation has

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focused on cycling or running exercise [4]. Despite the extremely high aerobic demands on

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cross-country skiers [22], and competitions often taking place in areas of elevated altitude, no

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previous studies have investigated the effects of NO3- supplementation on cross-country

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skiing exercise. The purpose of the present study was therefore to investigate the effects of an

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acute high dose of NO3- on submaximal and maximal cross-country skiing performance in

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competitive cross-country skiers exercising in laboratory-based normoxia and normobaric

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hypoxia. It was hypothesized that NO3- supplementation would increase plasma [NO3-] and

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[NO2-] compared to placebo, as well as lower the O2 cost of submaximal exercise and

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2. MATERIALS AND METHODS

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improve TT performance in hypoxia but not normoxia.

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2.1. Participants

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Eight well-trained cross-country skiers (5 males and 3 females, Table 1) competing at a

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national level participated in the current study, which took place during the competition

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season. Sample-size estimations were based on data reported by Lansley et al. [8] (n = 9) and

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12 participants were originally recruited to the study. Due to drop-out with symptoms of

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illness during the data collection period, eight participants completed the study, which is

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similar to several studies reporting the effects of NO3- supplementation on physiological

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function and exercise performance [6,11,23]. All eight participants were familiar with roller-

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skiing on a treadmill and had previously completed maximal treadmill roller-skiing tests as

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part of their regular monitoring. The participants were fully informed of the study procedures

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and their right to withdraw before providing written consent to participate. The study was

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ACCEPTED MANUSCRIPT 115

conducted in accordance to the declaration of Helsinki and approved by the Regional Ethical

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Review Board in Umeå, Sweden.

117 Table 1. Mean ± SD participant characteristics

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Age

Height

Body mass

V̇ O2max

V̇ O2max

(years)

(cm)

(kg)

(L·min-1)

(ml·kg-1·min-1)

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21.8 ± 2.8 178.8 ± 9.9 73.5 ± 8.3

5.23 ± 0.40 71.5 ± 4.7

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20.7 ± 1.2 171.6 ± 5.5 63.4 ± 5.7

3.69 ± 0.47 58.4 ± 2.5

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V̇ O2max: maximal oxygen uptake

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120 2.2. Study design

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Participants reported to the laboratory on five separate occasions. The first visit consisted of

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an incremental exercise test to exhaustion to determine maximal oxygen uptake (V̇ O2max), and

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familiarization of the experimental protocol. Visits 2–5 involved the experimental trials,

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which included each of the following: 1) exercise in normoxia (N) preceded by NO3--rich

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beetroot juice (BRJ) supplementation, 2) exercise in N preceded by NO3--deplete beetroot

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juice (PL) supplementation, 3) exercise in hypoxia (H) preceded by BRJ supplementation, 4)

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exercise in H preceded by PL supplementation. All exercise was performed on a treadmill

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(Rodby Innovation AB, Vänge, Sweden) using roller-skis (Pro-ski C2, Sterners Specialfabrik

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AB, Vansbro, Sweden) and the diagonal-stride technique for classical cross-country skiing.

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Experimental trials followed a randomised cross-over design and were double-blinded.

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2.3. Preliminary trial and familiarization

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Participants completed an incremental exercise test to exhaustion to determine V̇ O2max. The

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trial commenced with a 10-min warm-up at an incline of 6°, whereby the first 5 min was

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Thereafter, three 30-s high-intensity intervals were performed at a speed equal to the

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incremental test starting speed plus 4 km·h-1. The three intervals were separated by a 30-s

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recovery period, completed at the incremental test starting speed. The final 2.5 min of warm-

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up was performed at the incremental test starting speed plus 1 km·h-1. This warm-up was

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based on previous work conducted in the same laboratory [24]. Five minutes after the warm-

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up the test protocol commenced at an incline of 3° and a speed of 10, 11, or 12 km·h-1,

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depending on the sex, age and skiing ability of the participant. The incline of the treadmill

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was increased by 1° every minute until a maximum of 9°. Thereafter, speed was increased by

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0.4 km·h-1 each minute until exhaustion [25]. Respiratory variables were measured

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throughout exercise using a portable breath-by-breath device (Metamax 3B, Cortex, Leipzig,

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Germany), which was calibrated before every third test according to the manufacturer

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instructions. Heart rate (HR) was measured throughout exercise via telemetry (Polar H7,

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Kempele, Finland). V̇ O2max was accepted as the highest 30-s mean value and maximal HR

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was accepted as the highest value attained during the test. Participants rested for 15 minutes

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after the incremental exercise test, before being familiarized to the exercise protocol used

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during the four subsequent experimental trials.

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2.4. Experimental trials

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On the morning of the experimental trials participants consumed 140 ml of concentrated BRJ

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(~ 13 mmol NO3-) or PL (~ 0 mmol NO3-) (Beet It Sport, James White Drinks Ltd., Ipswich,

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UK) 2.5 hours prior to the start of the warm-up. Participants were asked to keep a record of

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their normal diet 24 hours prior to the first experimental trial, and to replicate this diet as

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closely as possible before each subsequent trial. Participants were also asked to refrain from

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alcohol and heavy exercise for 24 hours preceding testing. Caffeine was avoided on the

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morning before testing, and participants were instructed not to consume anything other than

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water after supplement ingestion. Each experimental trial was conducted at the same time of

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day for each individual, to minimise the influence of circadian variance, and a washout period

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of at least 72 hours followed BRJ supplementation.

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probes (Moxy muscle oxygen monitor, Fortiori design LLC, Minnesota, USA) were attached

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to the belly of m. vastus lateralis and m. triceps brachii on the left leg and arm, respectively,

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to enable assessment of the oxygenation status of specific muscle tissue involved in cross-

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country skiing [26,27]. The probe position was marked with indelible ink, to ensure identical

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probe placement across trials. Double-sided adhesive tape and elastic bandage were used to

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fasten the probes to prevent movement and extraneous light disturbing the measurement. The

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probes consisted of one emitter sending near-infrared light into muscle tissue, and two

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detectors. The ratio of O2-carrying haemoglobin to total haemoglobin, referred to as muscle

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oxygen saturation (SmO2), is reported as a percent.

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After attaching the NIRS probes, participants rested for 10 min in a seated position in

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normoxic conditions. During the 6th min of rest a baseline (BL) NIRS measurement was made

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and BP was measured after 7, 8 and 9 minutes of rest (Omron Intellisense M6, Omron

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healthcare Co. Ltd., Kyoto, Japan). Means from the two last BP measurements were used to

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determine systolic blood pressure (SBP) and diastolic blood pressure (DBP). After 10 min of

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seated rest a venous blood sample was drawn into a 4 mL lithium heparin tube (Vacutainer,

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Becton-Dickinson and company, New Jersey, USA). The blood sample was centrifuged

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immediately at 4000 rpm for 10 min at 4°C, after which plasma was extracted and

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immediately frozen at -80°C for later analysis of plasma [NO3-] and [NO2-], according to the

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ACCEPTED MANUSCRIPT method described by Hezel et al. . After blood sampling and five minutes prior to starting the

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warm-up participants entered the normobaric hypoxic chamber (HYPOXICO, Inc., New

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York, USA). The fraction of inspired oxygen (FIO2) was adjusted to correspond to the desired

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altitude for each trial, which was 16.8 ± 0.1% (~ 1800 m) during H and 20.9% (sea level)

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during N. Temperature was 18.0 ± 0.1 °C during the trials and relative humidity was 5.7 ±

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0.5% and 6.2 ± 0.6% in H and N, respectively.

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Participants completed a standardised 5-min warm up at an incline of 6° and a speed of 6, 6.5

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or 7.5 km·h-1, depending on sex, age and skiing ability. The warm up was followed by a 1-

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min rest period, before performing two, 6-min submaximal exercise bouts at an incline of 6°

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and a speed of 6.5, 7 or 8 km·h-1 for the first bout and 8, 8.5 or 9.5 km·h-1 for the second bout.

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The change in speed from the warm up to the first bout, and then from the first bout to the

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second bout, was equal to 0.5 km·h-1 and 1.5 km·h-1, respectively, for all individuals. One

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minute of rest separated the two submaximal exercise bouts, whereby the participants stood

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still on the treadmill. The speeds were chosen to elicit ~ 60% and 75% of V̇ O2max,

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respectively, and were based on speeds used by McGawley and Holmberg [25]. Submaximal

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values for V̇ O2, respiratory exchange ratio (RER) and HR were averaged over one minute

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between 4:30 and 5:30 during both submaximal bouts. A rating of perceived exertion (RPE)

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was recorded immediately after each submaximal bout using a 6–20-point scale [28] and

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SpO2 was measured using a fingertip pulse oximeter (Onyx Vantage, NONIN Medical Inc.,

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Minneapolis, USA). Blood samples were also collected immediately in 20 µl end-to-end

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capillary tubes for the subsequent analysis of [lactate] (BIOSEN S-Line, EKF Diagnostics,

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Cardiff, UK).

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ACCEPTED MANUSCRIPT Participants commenced the 1000-m TT after a 5-min rest period. Based on pilot testing and

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familiarization, the starting speed was 11.5 km·h-1 for females and 12.5 or 13.5 km·h-1 for

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males, depending on skiing ability. To regulate the speed during the TT the participants

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signalled to the test leader in control of the treadmill by raising their right hand to increase the

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speed and their left hand to decrease the speed. This form of signalling is a natural motion

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during diagonal skiing and occurs in time with the poling cycles. For each signal the speed

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changed by 0.3 km·h-1. Information regarding time and speed were concealed from the

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participants, but they were informed about elapsed distance every 200 m and verbally

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encouraged throughout the tests. Immediately following the TT, RPE was recorded, SpO2 was

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measured and a capillary blood sample was obtained after 2 minutes, as described above.

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Average values for V̇ O2, RER, and HR were determined from the entire TT, and maximal

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values were calculated over 30-s. Performance was measured as time to complete the TT

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(Timetot). After each trial participants were asked to report which environmental condition and

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supplement they believed they had received that day, in order to assess blinding success.

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2.5. Data analyses

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Analyses were performed using IBM SPSS Statistics Version 23 and data are presented as

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mean ± SD with a statistical significance level accepted at p ≤ 0.05. Normal distribution of

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data was determined by the Shapiro-Wilk’s test. Two-tailed paired sample t-tests were used to

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compare differences between BRJ and PL for resting SBP, DBP, plasma NO3- and NO2-

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concentrations, and SmO2. Variables associated with submaximal exercise were analyzed by

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two-way (altitude [N vs. H] x supplement [BRJ vs. PL]) repeated-measures ANOVA with

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Bonferroni corrections to reduce the risk of type I errors. Because not all participants

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managed to complete all four TTs, variables associated with TT performance were analyzed

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using two-tailed paired sample t-tests for BRJ vs. PL in N and H, separately. When

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ACCEPTED MANUSCRIPT appropriate, effect size was determined by Cohen’s d (calculated as the difference in means

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divided by the pooled SD) using the following definitions: trivial effect d < 0.2, small effect d

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≥ 0.2, moderate effect d ≥ 0.5, large effect ≥ 0.8 [29]. The 95% confidence interval (CI) for

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the difference between BRJ and PL in both N and H was calculated for the main performance

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variable (Timetot) using a publically available online spreadsheet [30]. NIRS-derived SmO2

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values were obtained at BL in normoxia, and during the last three minutes of both

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submaximal exercise bouts. NIRS data was not included from the TT, due to a number of

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missing data points from loss of signal.

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243 3. RESULTS

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3.1. Blinding success

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Participants successfully guessed the environmental condition (N or H) for 75% of the trials

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and incorrectly for 25% of the trials. In terms of supplementation, participants guessed

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correctly for 47% of the trials, incorrectly for 28% of the trials and did not know for 25% of

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the trials.

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3.2. Resting SmO2, blood pressure, plasma [NO3-] and [NO2-]

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There was no significant effect of supplement on resting SmO2 of the m. triceps brachii (BRJ:

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66 ± 11 vs. PL: 67 ± 6%, p = 0.669) or m. vastus lateralis (BRJ: 67 ± 7 vs. PL: 66 ± 6%, p =

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0.923). There was no significant effect of supplement on SBP (BRJ: 122 ± 10 vs. PL: 122 ± 9

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mmHg, n = 7, p > 0.999) or DBP (BRJ: 70 ± 6 vs. PL: 71 ± 6 mmHg, n = 7, p = 0.593).

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Conversely, plasma [NO3-] and [NO2-] were significantly elevated following BRJ compared to

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PL (588 ± 67 vs. 25 ± 5 uM for [NO3-]; 482 ± 188 vs. 129 ± 59 nM for [NO2-]; p < 0.001,

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Figure 1).

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Figure 1. (A.) Plasma nitrate (NO3-) and (B.) nitrite (NO2-) concentrations following placebo

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(PL) and beetroot juice (BRJ) supplementation.

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Significantly different from PL: * p < 0.001

ACCEPTED MANUSCRIPT 3.3. Submaximal exercise bout 1

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The first submaximal exercise bout was performed at 63 ± 4% of V̇ O2max (range: 54–70%).

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There was no significant effect of supplementation on V̇ O2, HR, RER, SpO2, RPE, [lactate],

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or SmO2 assessed at the m. vastus lateralis or m. triceps brachii (all p > 0.05, Table 2). By

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contrast, [lactate] was significantly higher (n = 7, p = 0.04), HR was significantly higher (p =

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0.012), SpO2 was significantly lower (p < 0.001), and SmO2 of the m. vastus lateralis was

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significantly lower (n = 7, p = 0.009) in H compared to N.

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3.4. Submaximal exercise bout 2

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The second submaximal exercise bout was performed at 75 ± 5% of V̇ O2max (range: 66–

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84%). Again, there was no significant effect of supplementation on VO2, HR, RER, SpO2,

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RPE, [lactate], or SmO2 assessed at the m. vastus lateralis or m. triceps brachii (all p > 0.05,

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Table 2). However, [lactate] and HR were significantly higher (p = 0.001 and p = 0.018,

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respectively) and SpO2 was significantly lower (p = 0.001) in H compared to N.

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Table 2. Mean ± SD responses to submaximal exercise in normoxia (N) and hypoxia (H) following beetroot juice (BRJ) and placebo (PL)

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supplementation

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ANOVA effects

BRJ-N

PL-N

BRJ-H

PL-H

Supplement

Altitude

Interaction

V̇ O2 (L·min-1)

8

2.92 ± 0.48

2.90 ± 0.49

2.94 ± 0.51

2.94 ± 0.53

p = 0.668

p = 0.609

p = 0.462

RER

8

0.90 ± 0.03

0.90 ± 0.04

0.91 ± 0.06

0.90 ± 0.04

p = 0.155

p = 0.763

p = 0.575

HR (beats·min )

8

151 ± 12

151 ± 15

159 ± 11

159 ± 10

p = 0.887

p = 0.012*

p = 0.885

SpO2 (%)

8

98 ± 1

98 ± 1

[lactate] (mmol·L-1)

7

1.54 ± 0.63

1.28 ± 0.23

RPE

8

12 ± 2

11 ± 1

SmO2 m. vastus lateralis (%)

7

50 ± 7

46 ± 6

SmO2 triceps brachii (%)

8

37 ± 14

39 ± 16

V̇ O2 (L·min-1)

8

3.48 ± 0.57

3.47 ± 0.55

RER

8

0.92 ± 0.03

HR (beats·min )

8

168 ± 9

SpO2 (%)

7

97 ± 1

[lactate] (mmol·L )

8

2.12 ± 0.90

RPE

8

SmO2 m. vastus lateralis (%) SmO2 triceps brachii (%)

88 ± 5

p = 0.789

p = 0.000***

p = 0.469

1.97 ± 0.45

1.68 ± 0.41

p = 0.228

p = 0.040*

p = 0.817

12 ± 1

12 ± 2

p = 0.072

p = 0.072

p = 0.692

42 ± 7

42 ± 5

p = 0.347

p = 0.009**

p = 0.366

33 ± 7

31 ± 10

p = 0.979

p = 0.229

p = 0.295

3.47 ± 0.54

3.50 ± 0.58

p = 0.769

p = 0.942

p = 0.495

0.92 ± 0.03

0.96 ± 0.06

0.94 ± 0.05

p = 0.343

p = 0.061

p = 0.454

168 ± 13

175 ± 9

176 ± 9

p = 0.958

p = 0.018*

p = 0.483

97 ± 1

87 ± 4

89 ± 5

p = 0.139

p = 0.001***

p = 0.455

1.82 ± 0.80

3.51 ± 1.05

3.51 ± 1.22

p = 0.524

p = 0.001***

p = 0.315

15 ± 1

13 ± 1

15 ± 1

14 ± 2

p = 0.076

p = 0.080

p = 0.197

7

41 ± 8

40 ± 9

36 ± 9

38 ± 15

p = 0.641

p = 0.143

p = 0.546

5

33 ± 16

44 ± 13

27 ± 12

24 ± 10

p = 0.319

p = 0.107

p = 0.093

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V̇ O2: oxygen uptake; RER: respiratory exchange ratio; HR: heart rate; SpO2: arterial oxygen saturation; [lactate]: blood lactate concentration;

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RPE: rating of perceived exertion; SmO2: muscle oxygen saturation

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All participants completed both TTs in N, whereas there were five uncompleted TTs in H (3

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in BRJ-H and 2 in PL-H), hence only four participants managed to complete all four TTs

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(Figure 2). Timetot was not significantly different between BRJ and PL in N (297 ± 29 s vs.

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295 ± 29 s, p = 0.216, 95% CI for the difference: -1.1 to 4.1) or H (305 ± 28 s vs. 301 ± 28 s,

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p = 0.358, 95% CI for the difference: -4.9 to 12.0). Three participants were marginally faster

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(1–3 s) with BRJ compared to PL in N, whereas only one participant was faster (5 s) with

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BRJ compared to PL in H. Supplementation with BRJ did not significantly affect average

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V̇ O2, HR or RER, maximal HR or RER, [lactate], SpO2 or RPE in N or H (all p > 0.05, Table

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3). In N, however, the maximal V̇ O2 was significantly lower following BRJ compared to PL

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(p = 0.039, Table 3) with the effect size considered to be trivial (d = 0.122). There was no

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difference in maximal V̇ O2 following BRJ and PL in H (p > 0.05, Table 3).

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Figure 2. 1000-m time-trial (TT) performance in (A.) normoxia and (B.) hypoxia following

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placebo (PL) and beetroot juice (BRJ) supplementation

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Table 3. Mean ± SD responses during the 1000-m TT in normoxia (N) and hypoxia (H) following beetroot juice (BRJ) and placebo (PL)

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supplementation

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PL-N

P value

Cohen’s d

BRJ-H

PL-H

P value

Cohen’s d

8

8

8

8

4

4

4

4

4.09 ± 0.81

4.17 ± 0.81

0.107

0.100

3.77± 0.78

3.72 ± 0.65

0.530

0.073

Maximal V̇ O2 (L·min )

4.56 ± 0.95

4.68 ± 0.97

0.039*

0.122

4.13 ± 0.87

4.15 ± 0.79

0.785

0.024

HRaverage (beats·min-1)

178 ± 5

178 ± 7

1.000

0.000

181 ± 7

180 ± 12

0.784

0.106

HRmax (beats·min )

188 ± 3

187 ± 6

0.456

0.189

189 ± 4

190 ± 6

0.406

0.291

RERaverage

1.03 ± 0.02

1.02 ± 0.03

0.502

1.06 ± 0.03

1.06 ± 0.04

0.897

10.75 ± 2.32

10.84 ± 1.71

0.901

a

a

-1

V̇ O2average (L·min ) -1

-1

RERmax -1

[lactate] (mmol·L )

94 ± 3

RPE

18 ± 2

96 ± 3

0.391

19 ± 1

0.205

1.05 ± 0.04

1.05 ± 0.04

1.000

0.000

0.038

1.10 ± 0.03

1.09 ± 0.07

0.690

0.180

13.20 ± 2.26

13.87 ± 1.39

0.354

b

b

0.045

a

0.641

0.584

0.209

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SpO2 (%)

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BRJ-N

a

88 ± 4

19 ± 1

89 ± 4 20 ± 1

0.225 0.391

0.356 b

0.260b 0.707

V̇ O2average: average oxygen consumption; Maximal V̇O2: maximal oxygen consumption; HRaverage: average heart rate; HRmax: maximal heart rate;

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RERaverage: average respiratory exchange ratio; RERmax: maximal respiratory exchange ratio; [lactate]: blood lactate concentration; SpO2: arterial

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oxygen saturation, RPE: rating of perceived exertion, : n = 4; : n = 3

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4. DISCUSSION

304 The principle original finding of the present study is that supplementation with an acute high

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dose of NO3- (∼ 13 mmol NO3-) does not significantly improve sport-specific physiological

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responses or exercise performance in competitive cross-country skiers exercising at sea level

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or simulated altitude (~ 1800 m). These results are consistent with previous findings showing

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no effects of NO3- supplementation on resting BP, submaximal or maximal exercise capacity

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in highly-trained individuals in N and H [9,12,13,31–33]. Importantly, these results occurred

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despite elevated plasma levels of NO metabolites after the ingestion of NO3--rich BRJ.

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A variety of NO3- supplementation strategies have led to lowered BP in both hypertensive and

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normotensive individuals [34], which was believed to depend on an increased bioavailability

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of NO causing vasodilation. Previous work using an acute high dose of ∼ 16.8 mmol NO3-

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reported significantly reduced SBP and DBP 2 h after ingestion, with a peak in plasma NO2-

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concentration and peak reduction of SBP occurring 4 h post supplementation [35]. The

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current findings, however, support those of Wilkerson et al. [11] and MacLeod et al. [32],

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who showed no effects of BRJ supplementation on BP in trained individuals. The conflicting

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findings might result from different amounts of ingested NO3-, the timing of BP

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measurements, or the training status of the participants. Indeed, NO3- supplementation did not

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affect mean arterial BP in elite cross-country skiers at rest or between submaximal and

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maximal exercise bouts, despite significantly elevated plasma levels of NO3- and NO2- [33].

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Since training itself might increase NOS activity [36–38], the effect of NO3- supplementation

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on BP may be reduced in athlete populations.

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ACCEPTED MANUSCRIPT Supplementation with BRJ in the present study did not affect any of the submaximal exercise

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variables or Timetot for the 1000-m TT in N or H. Interestingly, the maximal V̇ O2 obtained

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during the TT in N was significantly lower following BRJ compared to PL. Supplementation

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with NO3- has previously shown minor or no effects on V̇ O2max in healthy and trained

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individuals [4] and in general, a lowered V̇ O2max is associated with a reduced performance

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capacity [39]. However, NO3- supplementation reduced V̇ O2max during combined arm and leg

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cranking in healthy individuals [40] and reduced V̇ O2peak in cyclists [41], without

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compromising performance. The lowered maximal V̇ O2 during the TT could therefore be

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interpreted as an improved exercise efficiency, although this was not supported by any

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significant difference in V̇ O2average measured during TT following BRJ compared to PL, and

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maximal V̇ O2 was not lower with BRJ in H. However, the results regarding maximal V̇ O2

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following BRJ and PL in H might be affected by the small sample size (n = 4). According to

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Jones [38], a lowered V̇ O2max following NO3- is difficult to explain, since no adverse effects

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on cardiac function have been noted and would be rather unlikely. Therefore, further research

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appears necessary in order to better understand the mechanistic basis of any changes in

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V̇ O2max following NO3- supplementation.

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Individual responses to BRJ supplementation have previously been reported for cyclists [11],

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where five out of eight participants showed significantly increased plasma NO2- levels (>

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30%), as well as improved 50-mile TT performance, compared to PL. The other three

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participants had either lower (n = 1) or only marginally increased plasma NO2- levels (10%; n

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= 2) following BRJ compared to PL. Of these three so-called “non-responders”, only one

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participant improved their TT performance. In the current study, all eight participants showed

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an increase in plasma NO2- levels following BRJ compared to PL (by 59–88%), whereas only

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three participants reduced their Timetot in N (by 1 s [n = 2] or 3 s [n = 1]). In H, only one

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ACCEPTED MANUSCRIPT participant improved their TT performance following BRJ compared to PL (by 5 s), but this

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participant was not a “consistent responder”, since they were not one of the three who

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performed better following BRJ in N. Rather than characterising “responders” and “non-

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responders”, these small between-trial differences are more likely to reflect day-to-day

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variations in performance [42].

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While pre-supplementation baseline plasma NO3- and NO2- levels were not measured in the

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current study, previous studies using similar supplement products reported no significant

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differences in plasma NO3- or NO2- levels following PL compared to pre-supplementation

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baseline levels [11,20]. It may therefore be assumed that the NO3- and NO2- measured

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following ingestion of PL in the current study would not differ from the pre-supplementation

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baseline levels in the BRJ trials. The mean plasma level of NO2- following NO3-

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supplementation was slightly higher in the current study (482 ± 188 nM) compared to

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previous studies (325 ± 95 nM and 328 ± 107 nM) that have reported no effects on

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physiological responses to exercise or TT performance following NO3- supplementation in

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cross-country skiers [9,33]. These differences could be due to the higher dose of NO3- used in

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the current study (~ 13 mmol NO3- vs. ~ 10 mmol NO3-). Indeed, when supplemented with ~

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15 mmol NO3-, Shannon et al. [20] reported similar plasma NO2- levels as to those reported in

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the current study (468 ± 253 nM). However, these authors also reported a lower steady-state

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V̇ O2 and improved 1500-m running TT performance while exercising in moderate hypoxia (~

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2500 m). Therefore, plasma NO2- levels alone do not seem to explain the efficacy of

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supplementation in hypoxic conditions.

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For trained athletes who already possess a high performance capacity, even small

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improvements could be of importance [43]. Endurance training is known to increase the

ACCEPTED MANUSCRIPT amount of capillaries surrounding skeletal muscle fibers [44], thereby providing exercising

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muscles with a great O2 supply and increasing NOS activity [36,37], which may limit any

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additional effects of NO3- supplementation in trained individuals [45]. Athletes have also

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demonstrated higher baseline plasma NO3- and/or NO2- concentrations compared to lesser-

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trained individuals [11] which might be explained by a higher total calorie intake, a diet rich

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in NO3- [46] or improved NOS-activity caused by training itself [36,38]. It is therefore

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possible that the participants in the present study did not respond to NO3- supplementation due

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to their training status and/or diet. Alternatively, since the reproducibility of the specific TT

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test used in the current study has not been assessed, it is possible that real effects due to NO3-

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supplementation were masked by a lack of test sensitivity. However, this is unlikely since the

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intra-individual variation in performance across the trials was low. Moreover, TT tests of

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performance typically show very good reliability (i.e., CV < 4%), particularly when compared

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to open-ended tests to exhaustion [47,48].

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Compared to previous studies showing beneficial effects of NO3- supplementation in trained

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males exercising in H, the male participants in the present study had higher aerobic fitness

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levels and the females similar to the males in previous studies in terms of average V̇ O2max

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[17,19]. This adds further support to the argument that highly-trained individuals are less

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likely to benefit from NO3- supplementation, even after high doses (∼ 13 mmol NO3-).

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Muggeridge et al. [18] showed lowered resting BP and V̇ O2 during submaximal exercise in

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cyclists, as well as improved 16.1-km cycling TT performance, following a single ingestion of

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∼ 5 mmol NO3- in H. It is difficult to compare these participants to those included in the

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current study, since Muggeridge et al. [18] assessed V̇ O2peak values for their cyclists

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(described as competitive but non-elite athletes) at a simulated altitude of 2500 m, which

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would be expected to elicit lower peak values [49]. In the study by Shannon et al. [20],

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participants had an average V̇ O2max of ∼ 60 ml·kg-1·min-1 (range: ∼ 47–77 ml·kg-1·min-1), and

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all participants manifested a reduction in steady-state V̇ O2 and improvement in 1500-m TT

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performance assessed at a simulated altitude of 2500 m. Hence, a high dose of NO3- might

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still be beneficial for trained athletes exercising in moderate but not necessarily mild H.

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In order to mimic an altitude at which cross-country ski competitions are more realistically

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held, in comparison to previous studies that have typically simulated ≥ 2500 m, an O2 fraction

409

of 16.8% O2 was chosen to simulate an altitude of 1800 m in the present study. H alone led to

410

altered SpO2, HR and [lactate] responses during submaximal exercise, and possibly reduced

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performance capacity during the 1000-m TT, independent of BRJ supplementation. These

412

results are similar to those reported by MacLeod et al. [32] and are most likely explained by

413

the reduced O2 availability in H. Due to insufficient data obtained for the TTs in H, whereby

414

four subjects either fell or chose to abort the TT due to premature exhaustion, statistical

415

comparisons between N and H were not conducted. Since H affected HR, SpO2, [lactate], and

416

SmO2 at the m. vastus lateralis during submaximal exercise, it would be reasonable to expect

417

similar responses during the TTs. SpO2 in particular would be expected to be reduced during

418

high-intensity exercise in H, as seen in elite biathletes during maximal roller-skiing at an

419

altitude of ∼ 1800 m [50]. Indeed, the mean values for SpO2 and [lactate] presented in Table 3

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indicate a tendency to differ in H compared to N and a larger sample population may have

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revealed significant differences in these variables. The present study is limited by its small

422

sample size (n = 8), which is a recurring limitation when recruiting from athlete populations

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and was similar to other related studies [3,11,13].

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The present study evaluated the physiological and performance effects of acute rather than

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chronic NO3- supplementation.

This approach was based upon the rationale that acute

ACCEPTED MANUSCRIPT supplementation may be more willingly adopted by athletes, given the lower financial cost

428

and reduced time requirements compared with chronic supplementation [51]. Some previous

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research has indicated that chronic NO3- supplementation may be more effective than acute

430

NO3- supplementation [3,52], possibly because the physiological effects of NO3-, particularly

431

those involving changes in protein expression [53,54], may require multiple days of

432

supplementation to occur. In support of this notion, a recent meta-analysis observed a trend

433

towards greater effects of chronic compared with acute NO3- supplementation on exercise

434

TTE [4]. Furthermore, Kelly et al. [19] observed significant improvements in severe-intensity

435

exercise tolerance in H following 3 days of NO3- supplementation. Therefore, it is possible

436

that greater physiological and/or performance effects may have been observed in the present

437

study if participants were supplemented chronically with NO3-.

438

Muggeridge at al. [18] and Shannon et al. [20] observed significant improvements in TT

439

performance in H following acute NO3- supplementation strategies. Several previous studies

440

in N have also reported improved exercise performance following acute NO3-

441

supplementation [8,23,55]. This suggests that chronic loading is not essential to elicit a

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beneficial effect of NO3- supplementation, and that the lack of effect of NO3- on exercise

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performance in the current study cannot entirely be attributed to the acute supplementation

444

strategy employed.

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Prior to the TT in the present study participants completed two constant-load exercise bouts at

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speeds eliciting ~ 60% and 75% of normoxic V̇ O2max, based on speeds used by McGawley

448

and Holmberg [25]. It should be noted that matching absolute exercise intensities leads to

449

different physiological responses in normoxia and hypoxia, which may therefore result in

450

athletes exercising within different exercise-intensity domains in the two environments [56].

451

Furthermore, muscle recruitment is altered in normoxia and hypoxia when absolute work

ACCEPTED MANUSCRIPT rates are matched [57,58], which may influence the efficacy of NO3- supplementation due to

453

different effects of NO3- as the degree of type II muscle activation changes [23,54,59,60].

454

While the matching of absolute work rates may be considered a limitation of the current study

455

design, the key aim was not to evaluate the effects of NO3- supplementation in relation to any

456

specific exercise-intensity domain. Instead, the submaximal speeds were selected to enable

457

evaluation of the effects of NO3- supplementation at two distinct work rates, both of which

458

could be performed effectively using the diagonal skiing sub-technique in normoxia and

459

hypoxia. A second limitation of the study may be the use of the MOXY NIRS device. This

460

device has been used in several previous studies to non-invasively monitor muscle tissue

461

oxygenation during exercise, including cross-country skiing [27], and SmO2 measurements

462

have been shown to be reliable during low- and moderate-intensity cycle ergometry [61].

463

However, reliability during cross-country skiing and the validity of this device compared with

464

other NIRS devices remains unclear. The NIRS signal can be influenced by several factors,

465

including adipose tissue thickness, which dampens the NIRS signal [62,63], skin blood flow,

466

which is elevated during exercise for thermoregulation [64], and motion artefacts [61]. Thus,

467

further studies addressing the validity of this device and reliability during whole-body

468

exercise may be warranted.

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469 4.1. Conclusion

471

Despite elevated plasma levels of NO metabolites, the cross-country skiers in the present

472

study did not benefit from an acute high dose of NO3- (∼ 13 mmol) in terms of submaximal

473

exercise variables or maximal exercise performance in either N or moderate H.

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474 475

CONFLICT OF INTEREST AND SOURCE OF FOUNDING

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EW is a co-director of Heartbeet, a company that seeks to identify therapeutic potential of

477

dietary nitrate. This research did not receive any specific grant from funding agencies in the

478

public, commercial, or not-for-profit sectors.

479 ACKNOWLEDGMENTS

481

The authors thank the participants for their time and commitment to the study, despite the

482

ongoing competition season. The authors also thank Dr Helen Hanstock, Alexander Patrician,

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Andreas Kårström and Hampus Lindblom for assistance and guidance regarding equipment

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and sampling methods.

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ACCEPTED MANUSCRIPT 485

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ACCEPTED MANUSCRIPT [23] O.M. Shannon, M.J. Barlow, L. Duckworth, E. Williams, G. Wort, D. Woods, M. Siervo, J.P. O’Hara, Dietary nitrate supplementation enhances short but not longer duration running time-trial performance, Eur. J. Appl. Physiol. 117 (2017) 775–785. doi:10.1007/s00421-0173580-6.

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[24] J. Watkins, S. Platt, E. Andersson, K. McGawley, Pacing strategies and metabolic responses during 4-minute running time-trials, Int. J. Sports Physiol. Perform. (2017) 1–24. doi:10.1123/ijspp.2016-0341. [25] K. McGawley, H.-C. Holmberg, Aerobic and anaerobic contributions to energy production among junior male and female cross-country skiers during diagonal skiing, Int. J. Sports Physiol. Perform. 9 (2014) 32–40. doi:10.1123/ijspp.2013-0239.

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ACCEPTED MANUSCRIPT HIGHLIGHTS

Physiological and performance effects of nitrate supplementation during roller-skiing in normoxia and normobaric hypoxia

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Authors: Linn Nybäck, Caroline Glännerud, Gustav Larsson, Eddie Weitzberg, Oliver Michael Shannon, Kerry McGawley.

Highlights

13 mmol NO3- significantly elevated plasma NO3- and NO2- levels in trained athletes



However, there was no benefit of acute NO3- supplementation in normoxia or hypoxia



Physiological responses to submaximal exercise were not altered by NO3- ingestion



Neither was roller-skiing time-trial performance improved with NO3- supplementation

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