Neither Beetroot Juice Supplementation nor Increased Carbohydrate Oxidation Enhance Economy of Prolonged Exercise in Elite Race Walkers

ven the importance of exercise economy to endurance performance, we implemented two strategies purported to reduce the oxygen cost of exercise within a 4 week training camp in 21 elite male race walkers. Fourteen athletes undertook a crossover investigation with beetroot juice (BRJ) or placebo (PLA) [2 d preload, 2 h pre-exercise + 35 min during exercise] during a 26 km race walking at speeds simulating competitive events. Separately, 19 athletes undertook a parallel group investigation of a multi-pronged strategy (MAX;n= 9) involving chronic (2 w high carbohydrate [CHO] diet + gut training) and acute (CHO loading + 90 g/h CHO during exercise) strategies to promote endogenous and exogenous CHO availability, compared with strategies re ecting lower ranges of current guidelines (CON;n= 10). There were no differences between BRJ and PLA trials for rates of CHO (p= 0.203) or fat (p= 0.818) oxidation or oxygen consumption (p= 0.090). Compared with CON, MAX was associated with higher rates of CHO oxidation during exercise, with increased exogenous CHO use (CON; peak = ~0.45 g/min; MAX: peak = ~1.45 g/min,p< 0.001). High rates of exogenous CHO use were achieved prior to gut training, without further improvement, suggesting that elite athletes already optimise intestinal CHO absorption via habitual practices. No differences in exercise economy were detected despite small differences in substrate use. Future studies should investigate the impact of these strategies on sub-elite

= ~0.45 g/min; MAX: peak = ~1.45 g/min,p< 0.001). High rates of exogenous CHO use were achieved prior to gut training, without further improvement, suggesting that elite athletes already optimise intestinal CHO absorption via habitual practices. No differences in exercise economy were detected despite small differences in substrate use. Future studies should investigate the impact of these strategies on sub-elite athletes' economy as well as the performance effects in elite groups. Keywords:exercise fuel; gut training; exogenous CHO; CHO loading; endurance sport; sucralose Nutrients2021,13, 2767.

Nutrients2021,13, 2767 2 of 22 1. Introduction Success in endurance events is underpinned by sustained or periodic achievement of high speed/power outputs which represent high relative and absolute exercise intensities, and are linked to an interaction between the athlete's maximal aerobic power ( . V O 2peak), the fraction of . V O 2peakthat can be sustained for the event distance, and the oxygen (O2) cost of movement (e.g., running/walking economy) [1–5]. Training and nutrition strategies for endurance performance aim to enhance various aspects of these characteristics [6], includ- ing ensuring that suitable substrates are able to fuel the event over its entire duration [4,7]. The economy of running or walking represents the relationship between oxygen utilisation and speed of locomotion [8], with a higher economy (lower oxygen cost for a given speed) at event-speci c speeds being a better predictor of performance among a group of sub- elite/elite runners than . V O 2peak[9]. A range of training, environmental and biomechanical factors are known to affect running economy [10] and the recent breaking of the two-hour marathon barrier has renewed interest in economy as a determinant of endurance per- formance [11]. Indeed, testimonials [12] and studies related to new designs of running shoes [13] and the design of pacemaker formations to minimise air resistance for a targeted runner [14,15] have contributed to the achievement of this feat via the enhancement of running economy. Several nutritional factors are also known to affect the economy of locomotion. Indeed, the popularity of beetroot juice supplements, used by athletes as a source of dietary nitrate, stems from early observations of a reduction in the oxygen cost of submaximal cycling [16,17] and running [18] following nitrate supplementation. Dietary nitrate can be serially reduced to nitric oxide (NO), providing a complementary pathway to the nitric oxide synthase (NOS-)supported conversion of arginine to NO [19]. The nitrate- NO pathway is of particular importance under the hypoxic and acidic conditions often occurring locally in the exercising muscle. In addition to enhancing oxygen delivery to the muscle or to speci c bres, nitrate supplementation is thought to reduce

nitric oxide (NO), providing a complementary pathway to the nitric oxide synthase (NOS-)supported conversion of arginine to NO [19]. The nitrate- NO pathway is of particular importance under the hypoxic and acidic conditions often occurring locally in the exercising muscle. In addition to enhancing oxygen delivery to the muscle or to speci c bres, nitrate supplementation is thought to reduce the oxygen cost of exercise via enhanced calcium handling and contractile ef ciency [19], while earlier suggestions of enhanced mitochondrial ef ciency are now disputed [20,21]. Although nitrate supplementation is more likely to bene t higher-intensity events [19], speci c bene ts to longer endurance events might occur via an attenuation of the gradual rise in oxygen cost during prolonged exercise [22] or enhanced . V O2kinetics in the transition to a higher speed [23] as occurs when athletes change pace at critical times within a race. Previous studies of nitrate supplementation during prolonged exercise may have failed to detect bene ts if the typical protocol of nitrate ingestion ~2 h pre-event was associated with gradual reduction in plasma nitrite concentrations before the end of the protocol [19]. Hence, supplementation protocols for endurance events might warrant nitrate intake before andduringthe event to maintain elevated nitrite concentrations [22]. Differences in exercise economy due to the choice of muscle substrate have also received recent attention following consistent ndings from studies from our group [24–27] and others [28] that the substantial increase in muscle fat oxidation achieved by adaptation to a ketogenic low-carbohydrate (CHO), high-fat diet is associated with an increase in the oxygen cost of exercise at speeds which are relevant to the race performance of elite endurance athletes [29]. Indeed, as demonstrated more than a century ago [30,31], and explained by the stoichiometry of oxidative reactions [32], CHO oxidation produces 5–8% higher energy yield per litre of oxygen consumed through oxidative phosphorylation. Although contemporary sports nutrition guidelines for endurance performance already promote strategies to match CHO availability to the fuel demands of the event [7,33], it is worth considering whether further, even subtle, increases in CHO utilisation during the event might

the stoichiometry of oxidative reactions [32], CHO oxidation produces 5–8% higher energy yield per litre of oxygen consumed through oxidative phosphorylation. Although contemporary sports nutrition guidelines for endurance performance already promote strategies to match CHO availability to the fuel demands of the event [7,33], it is worth considering whether further, even subtle, increases in CHO utilisation during the event might enhance economy in a meaningful way; either allowing the athlete to increase their speed for the same oxygen utilisation or reducing the oxygen and metabolic cost of a given speed. For example, according to our modelling [4], a 55 kg marathon runner with a sustainable . V O2of 3.75 L/min and energy cost of 180 mL/kg/km would achieve a

Nutrients2021,13, 2767 3 of 22 sustainable marathon running speed of 20.83 km/hr, with a nishing time of 2:01:33. In this scenario, a 0.05 increase in respiratory quotient (e.g., from 0.85 to 0.90) would induce a ~0.9% increase in the energy liberated per L of oxygen consumed, translating into a similar improvement in running speed (to 21.02 km/hr) and a 66 s improvement in nishing time (2:00:27). Whether theoretical calculations like these translate into real-life performance improvements, and whether CHO availability/utilisation can be further increased above the rates already achieved by elite endurance athletes needs to be investigated. Carbohydrate utilisation during prolonged endurance exercise is in uenced by numer- ous factors, including the intensity and duration of the exercise, environmental conditions and the availability of endogenous and exogenous CHO (for reviews, see [34,35]). While muscle glycogen stores can be supercompensated by diet and training protocols to increase their contribution to muscle substrate use [36], the rate of oxidation of exogenous CHO from sources consumed prior to and during exercise is affected by the type and amount of CHO intake, and by prior adaptation of the gut via repeated exposure to enhance tol- erance and intestinal absorption [37–39]. Although evidence for this `training' effect has only been investigated using glucose (demonstrating upregulation of its transport protein, SGLT1), fructose absorption via GLUT5 is also known to upregulate rapidly following CHO exposure [39]. Additionally, the ingestion of arti cial sweeteners such as sucralose can also increase SGLT1 content in animal models [40] via a cascade initiated by sweet taste receptors in the mouth and small intestine. Together, these strategies may enhance CHO availability and oxidation during prolonged high-intensity endurance exercise, leading to an improvement in exercise economy. Accordingly, the aim of this project was to undertake separate investigations of two different dietary approaches to enhance exercise economy in elite athletes during pro- longed exercise simulating race pace in endurance (race walking) events. The rst strategy (Beetroot Juice; BRJ) involved nitrate supplementation, using a newly modi ed protocol suited to endurance sports: a 2 d pre-load combined with an acute BRJ protocol providing a total

to undertake separate investigations of two different dietary approaches to enhance exercise economy in elite athletes during pro- longed exercise simulating race pace in endurance (race walking) events. The rst strategy (Beetroot Juice; BRJ) involved nitrate supplementation, using a newly modi ed protocol suited to endurance sports: a 2 d pre-load combined with an acute BRJ protocol providing a total dose (~19 mmol) known to achieve a physiological effect [41], but consumed before and during exercise [22]. The second strategy (Carb Max) was a multi-pronged protocol involving chronic diet-training adaptations and acute race strategies to maximise oxidation of endogenous and exogenous CHO sources during exercise. Our comparison (control) condition involved current sports nutrition guidelines at the “low end” of recommended CHO intake ranges [42], since this often represents real life practice [43,44]. Although we recognised that the multiple treatments in our second strategy would prevent us from identifying the speci c contribution of any single element, we considered this to be a proof-of-concept study in which the likelihood of detecting changes to substrate use and exercise economy were highest. If such an outcome was achieved, it would then merit separate investigation of the individual components. 2. Materials and Methods 2.1. Overview and Participants Twenty-one elite male race walkers participated in one of two training camps (Jan- uary or May) held at the Australian Institute of Sport (AIS). The cohort, which included 16 athletes of high-level international representation (e.g., Olympic Games, World Champi- onships, World Walking Cup) and ve national level training partners, were recruited via targeted invitations from key athletes and the coach with whom the study was planned. This study conformed to the standards set by theDeclaration of Helsinkiand was approved by the Human Research Ethics Committee of the AIS (no 20171203). All subjects provided informed consent after being provided with study details both verbally and in writing. During the training camps, held over a ~4 week duration, athletes were accommodated at the residential facilities at the AIS to allow all training to be supervised and to permit strict dietary control for the various study interventions [45]. Participants had

AIS (no 20171203). All subjects provided informed consent after being provided with study details both verbally and in writing. During the training camps, held over a ~4 week duration, athletes were accommodated at the residential facilities at the AIS to allow all training to be supervised and to permit strict dietary control for the various study interventions [45]. Participants had been undertaking base phase endurance training prior to the training camp, and the weekly training program (typically, 90–140 km/week) represented an intensi ed training block prior to early season

Nutrients2021,13, 2767 4 of 22 competition. It consisted of a number of compulsory group race walking sessions, some sessions undertaken as intervention trials and the opportunity for athletes to complete additional sessions of race walking, gym and cross-training. The 4 week schedule was arranged to accommodate the investigation of two separate dietary interventions for exercise economy: Study 1: Beetroot Juice study and Study 2: Carb Max (see Figure summary of protocol). Overall, 15 athletes attended the rst camp, while an additional 6 athletes completed the second. Figure number of participants who completed each investigation. Characteristics of athletes who contributed to each part of the project are summarised in Table.Nutrients 2021, 13, x FOR PEER REVIEW 4 of 22 During the training camps, held over a ~4 week duration, athletes were accommo- dated at the residential facilities at the AIS to allow all training to be supervised and to permit strict dietary control for the various study interventions [45]. Participants had been undertaking base phase endurance training prior to the training camp, and the weekly training program (typically, 90–140 km/week) represented an intensified training block prior to early season competition. It consisted of a number of compulsory group race walking sessions, some sessions undertaken as intervention trials and the opportunity for athletes to complete additional sessions of race walking, gym and cross-training. The 4 week schedule was arranged to accommodate the investigation of two separate dietary interventions for exercise economy: Study 1: Beetroot Juice study and Study 2: Carb Max (see Figure 1 for summary of protocol). Overall, 15 athletes attended the first camp, while an additional 6 athletes completed the second. Figure 1 summarises the design of each study and the number of participants who completed each investigation. Characteristics of athletes who contributed to each part of the project are summarised in Table 1. Figure 1. Schematic of the two investigations (Beetroot Juice: Figure 1A and Carb Max: Figure 1B) scheduled within a 4 week training camp involving 21 elite male race walkers. BM: body mass; BRJ: beetroot juice; CHO: carbohydrate; CON: control condition; low res: low residue/fibre diet; MAX:

athletes who contributed to each part of the project are summarised in Table 1. Figure 1. Schematic of the two investigations (Beetroot Juice: Figure 1A and Carb Max: Figure 1B) scheduled within a 4 week training camp involving 21 elite male race walkers. BM: body mass; BRJ: beetroot juice; CHO: carbohydrate; CON: control condition; low res: low residue/fibre diet; MAX: Carb Max intervention. Table 1. Characteristics of 21 elite male race walkers who participated in this project. Study One: BRJ Study Two: Carb Max Characteristics ( n = 14) CON ( n = 10) MAX ( n = 9) Age (y) 30.7 (4.2) 29.4 (4.6) 29.7 (4.2) Body Mass (kg) 67.9 (4.7) 68.4 (9.4) 68.7 (5.0) í µí±‰ 6O2peak (mL/kg/min) 63.9 (5.5) 60.9 (5.3) 63.1 (4.6) 10 km personal best (min:sec.00) 40:45.32 (1:02.50) 41:11.21 (1:33.31) 40:55.00 (1:03.36) 20 km personal best (hr:min.sec) 1:22.53 (0:02.03) 1:24.29 (0:04.45) 1:23.04 (0:01.59) Data are reported as mean (standard deviation). 2.2. Overview of Dietary Control and Interventions Figure 1. Schematic of the two investigations (Beetroot Juice: FigureA and Carb Max: FigureB) scheduled within a 4 week training camp involving 21 elite male race walkers. BM: body mass; BRJ: beetroot juice; CHO: carbohydrate; CON: control condition; low res: low residue/ bre diet; MAX: Carb Max intervention. Table 1.Characteristics of 21 elite male race walkers who participated in this project. Study One: BRJ Study Two: Carb Max Characteristics ( n= 14) CON ( n= 10) MAX ( n= 9) Age (y) 30.7 (4.2) 29.4 (4.6) 29.7 (4.2) Body Mass (kg) 67.9 (4.7) 68.4 (9.4) 68.7 (5.0) . VO 2peak (mL/kg/min) 63.9 (5.5) 60.9 (5.3) 63.1 (4.6) 10 km personal best min:sec.00) 40:45.32 (1:02.50) 41:11.21 (1:33.31) 40:55.00 (1:03.36) 20 km personal best (hr:min.sec) 1:22.53 (0:02.03) 1:24.29 (0:04.45) 1:23.04 (0:01.59) Data are reported as mean (standard deviation). 2.2. Overview of Dietary Control and Interventions Dietary treatments provided in this project were implemented using methods previ- ously described elsewhere in detail [45]. Brie y, all foods and uids consumed during the study were prescribed and provided by a team of chefs, food service dietitian and sports dietitians. Menus were constructed

(0:01.59) Data are reported as mean (standard deviation). 2.2. Overview of Dietary Control and Interventions Dietary treatments provided in this project were implemented using methods previ- ously described elsewhere in detail [45]. Brie y, all foods and uids consumed during the study were prescribed and provided by a team of chefs, food service dietitian and sports dietitians. Menus were constructed for the various phases of each study, and then meal plans were individually developed for each athlete to integrate personal food preferences

The research explores dietary interventions to enhance exercise economy in elite race walkers.