Experimental Protocol

To control dietary intake, all foods and fluids were provided throughout the experimental trials, and participants were required to consume a standardized low fermentable carbohydrate (FODMAP) diet during the 24 h before, and throughout the experimental trials. Meals were designed in accordance with current nutrition guidelines for endurance athletes, and calculated to provide <2 g FODMAP per meal using a FODMAP specific database (Monash University, FoodWorks Professional 7, Xyris, Brisbane, Australia) (Thomas et al., 2016; Gaskell et al., 2020b). Compliance was assessed using a food and fluid diary [overall mean (SD): energy 10.1 (3.0) MJ/day, protein 98 (30) g/day, fat 57 (36) g/day, carbohydrate 353 (87) g/day, fiber 44 (11) g/day, and water 2,333 (1,358) ml/day]. Participants were asked to avoid alcohol and strenuous exercise during the 48 h before each experimental trial, and to refrain from consuming caffeinated beverages during the 24 h before each experimental trial. In a randomized order (computer generated randomization), generated by an independent third-party researcher, participants completed two experimental trials separated with at least a 5 days washout period, to accommodate the participants’ availability. Trials for female athletes were scheduled during the follicular phase of their menstrual cycle (n = 5) or when taking the active medication of the oral contraceptive pill (n = 1). Resting estrogen levels (DKO003/RUO; DiaMetra, Italy) were measured for verification, were within normal reference range, and did not differ between trials [11.6 (6.0) pg/ml; P = 0.593] (Snipe and Costa, 2018b). Participants reported to the laboratory at 0800 h after consuming the standardized mixed carbohydrate breakfast [energy 2.9 (0.8) MJ, protein 28 (9) g, fat 19 (5) g, carbohydrate 99 (28) g, fiber 12 (5) g, and water 363 (264) ml] at ∼0700 h. Before commencing the exercise protocol, participants were asked to void. Pre-exercise nude BM and total body water (TBW) (Seca 515 MBCA, Seca Group, Hamburg, Germany) were recorded. Participants inserted a thermocouple 12 cm beyond the external anal sphincter to record pre-exercise rectal temperature (T_re) (Precision Temperature 4600 Thermometer, Alpha Technics, CA, United States). Participants provided a breath sample into a 250 ml breath collection bag (Wagner Analysen Technik, Bremen, Germany), and completed an exercise-specific mVAS gastrointestinal symptom (GIS) assessment tool (Gaskell et al., 2019). Blood was collected by venepuncture from an antecubital vein into three separate vacutainers (6 ml 1.5 IU/ml lithium heparin, 4 ml 1.6 mg/ml K₃EDTA, and 5 ml SST; BD, Oxford, United Kingdom). The exercise protocol consisted of a 2 h (initiated at 0900 h) high intensity interval running exercise (HIIT) session in T_amb 23.4 (1.1)°C and 44 (6)% RH, as described in Figure 1. The protocol was designed to provide sufficient exercise stress to perturb key markers of physiological and metabolic homeostasis (e.g., muscle glycogen, muscle protein, and hydration), including immune and gastrointestinal status, as previously reported (Snipe et al., 2018a; Costa et al., 2019, 2020b; Russo et al., 2019), and to mimic exertional strain incurred by continuous and intermittent endurance activities (e.g., recreational or competitive). During exercise, participants were provided with water equivalent to 3 ml/kg BM/h (Costa et al., 2009, 2011). HR (Polar Electro, Kempele, Finland), RPE, and thermal comfort rating (TCR) were measured at the 15 min mark of each 20 min cycle (Costa et al., 2014). Recovered HR and GIS were measured during the final 30 s of the 20 min cycle. Immediately post-exercise, nude BM and T_re were recorded. The recovery period commenced 30 min after the end of the exercise protocol to prepare for muscle biopsy sampling. Participants rested in a supine position in a sterile phlebotomy room for venous blood sampling followed by the first muscle biopsy thereafter. Muscle biopsy samples were taken 0 and 2 h into the recovery period. TBW was measured immediately after muscle biopsy sampling. Blood samples, nude BM and TBW were collected again at 2 and 4 h of recovery. Breath samples were collected and GIS recorded every 30 min throughout the recovery period. Total urine output was collected throughout the total recovery period. Weight of urine output was recorded at 2 and 4 h of recovery. After sampling at 2 h post-exercise, participants received a standardized recovery meal [energy 2.8 (0.7) MJ, protein 31 (8) g, fat 4 (2) g, carbohydrate 137 (32) g, fiber 9 (2) g, and water 415 (103) ml], and were instructed to consume as much as tolerable, and the total weight of the meal consumed was recorded. In addition, participants consumed a standardized evening meal after leaving the laboratory [energy 3.1 (1.4) MJ, protein 32 (14) g, fat 18 (17) g, carbohydrate 102 (50) g, fiber 19 (6) g, and water 757 (52) ml].

Schematic illustration of the experimental design. NBM, nude body mass; TBW, total body water; VBS, venous blood sampling; UO, urine output and osmolality; BH₂, breath hydrogen; GIS, gastrointestinal symptoms; HR, heart rate; TCR, thermal comfort rating; RPE, rating of perceived exertion; MB, muscle biopsy; CBS, capillary blood sampling; RTIME, readiness to invest mental effort; RTIPE, readiness to invest physical effort; RER, respiratory exchange ratio; CM, chocolate flavored dairy milk; CEB, carbohydrate-electrolyte beverage; BM, body mass.

The following morning, participants returned to the laboratory (0800 h) to assess psychophysiological parameters and exercise performance. Due to unforeseen circumstances unrelated to the study intervention, 3 participants did not return for the second day of testing on one or both of their trials. Therefore, data for 14 participants (n = 9 males, n = 5 females) was included for analysis. A standardized mixed carbohydrate breakfast [energy 2.9 (1.0) MJ, protein 29 (12) g, fat 18 (6) g, carbohydrate 97 (37) g, fiber 11 (5) g, and water 414 (235) ml] was consumed at 0700 h. Nude body mass, TBW and GIS were recorded on arrival and again after the performance test. Before and after the performance test, participants completed measures of readiness to invest mental and physical effort, rated from 0 to 10, with higher ratings indicating greater readiness to invest effort (Duncan et al., 2012). Participants performed a 20 min running exercise bout to measure oxygen uptake and oxidation rates at four submaximal exercise intensities (50, 60, 70, and 80% V̇O_2max) for 5 min each. Thereafter, in accordance with the cohort population (recreationally trained endurance athletes and varied endurance modalities) they were asked to complete a 1 h performance test in thermoneutral conditions [T_amb 23.0 (1)°C and 46 (9)% RH]. Participants were instructed to run the maximal distance they were capable of running in 1 h, with the incline set at 1%, as previously reported (Oliver et al., 2007, 2009; Costa et al., 2017a). Total distance, HR, RPE, and water intake (provided ad libitum) were recorded every 10 min.