Heat Acclimation Following Heat Acclimatization Elicits Additional Physiological Improvements in Male Endurance Athletes

he purpose of this study was to assess the effectiveness of heat acclimatization (HAz) followed by heat acclimation (HA) on physiological adaptations. 25 male endurance athletes (age 36 12 y , height 178.8 6.39 cm, body mass 73.03 8.97 kg, and VO 2peak57.5 7.0 mL kg 1 min 1 ) completed HAz and HA. HAz was 3 months of self-directed summer training. In the laboratory, a5-day HA prescribed exercise to target a hyperthermic zone (HZHA) of Trecbetween38.50 and 39.75 C for 60 min. Exercise trials were 60 min of running (59% 2% VO 2peak) in an environmental chamber (wet bulb globe temperature 29.53 0.63 C)

kg 1 min 1 ) completed HAz and HA. HAz was 3 months of self-directed summer training. In the laboratory, a5-day HA prescribed exercise to target a hyperthermic zone (HZHA) of Trecbetween38.50 and 39.75 C for 60 min. Exercise trials were 60 min of running (59% 2% VO 2peak) in an environmental chamber (wet bulb globe temperature 29.53 0.63 C) and administered at: baseline, post-HAz, and post-HAz+HA. Measured variables included internal body temperature (Trec), heart rate (HR), and sweat rate (SR). Repeated measure ANOVAs and post hoc comparisons were used to assess statistically signi cant (p< 0.05) differences. Trecwas lower post-HAz+HA (38.03 0.39 C) than post-HAz (38.25 0.42 C, p= 0.009) and baseline (38.29 0.37 C,p= 0.005). There were no differences between baseline and post-HAz (p= 0.479) in Trec. HR was lower post-HAz (143 12 bpm,p= 0.002) and post-HAz+HA (134 11 bpm,p< 0.001) than baseline (138 14 bpm). HR was lower post-HAz+HA than post-HAz (p= 0.013). SR was higher post-HAz+HA (1.93 0.47 L h 1 ) than post-HAz (1.76 0.43 L h 1 , p= 0.027). Combination HAz and HA increased physiological outcomes above HAz. This method can be used to improve performance and safety in addition to HAz alone. Keywords: thermoregulation; heat mitigation; aerobic; heat tolerance; heat illness; training strategy 1. Introduction Athletes from around the world travel to compete in hot weather conditions of which they are not accustomed to [1]. Heat acclimatization (HAz) is a heat mitigation strategy that is often proposed to athletes and coaches as a means of optimizing performance and preventing heat illness. The idea of HAz is simple: train outside in the heat to adapt. The implementation of this idea with athletes, however, is not as simple to effectively implement. For example, regional differences in environmental conditions, such as the mild summers experienced by many across the globe, make it dif cult for athletes who are traveling to hotter venues to gain the full bene ts of HAz needed prior to competition [2]. One solution to this problem is the use of heat acclimation (HA), which is the sys- tematic process of repeated

differences in environmental conditions, such as the mild summers experienced by many across the globe, make it dif cult for athletes who are traveling to hotter venues to gain the full bene ts of HAz needed prior to competition [2]. One solution to this problem is the use of heat acclimation (HA), which is the sys- tematic process of repeated exposures to a thermally extreme environment that elicits positive physiological and perceptual adaptations [3] in an arti cial environment. Typical responses following HA include lower heart rate (HR), internal body temperature, skin Int. J. Environ. Res. Public Health2021,18, 4366.

Int. J. Environ. Res. Public Health2021,18, 4366 2 of 12 temperature (T sk), and sweat electrolyte concentration, and increased plasma volume and sweat rate (SR) [4]. The time course of HA adaptions are well-established, with SR typically being the adaptation that occurs at least 7–10 days into HA [4–7]. While this strategy is an effective solution, the cost and time constraints that are often placed on athletes and coaches make the recommended 7–14 day protocols dif cult to achieve. There is no consensus on a universal, optimal protocol [7] for HA, HAz, or a combination of the two. One way in which HAz may be advantageous to HA in most environmental labo- ratories is that HAz introduces radiant heat from the sun as a stimulus for physiological adaptation, but HAz may not provide an adequate thermal load (depending on training location and environmental conditions) for optimal thermoregulatory adaptations [8]. HA may produce greater physiological and performance outcomes than HAz, however, it can be dif cult to balance normal sport training with an intense HA protocol for a long period of time [3]. Even amongst HA protocols, various methods have been utilized [7,9] and no consensus has been made as to the “optimal” approach. The xed-work rate method, that involves a steady exercise intensity, can elicit a thermal stress through an elevated internal body temperature. However, one limitation to this method is that if the workload is intense enough to drive internal body temperature up, the session must stop when the participant experiences volitional fatigue or the laboratory temperature cut-off point is reached, potentially limiting the duration that an individual is exercising in these conditions. Alternatively, if the intensity is too low, the drive to reach an elevated internal body temperature will lead to a long session duration, which is often not feasible for athletes when attempting to maintain an appropriate training balance [10]. Self- paced HA has also been examined in previous research, however, this method allows the athlete to self-select the intensity, which could result in a reduced thermal, cardiovascular, and relative work-load [7]. One of the most popular

will lead to a long session duration, which is often not feasible for athletes when attempting to maintain an appropriate training balance [10]. Self- paced HA has also been examined in previous research, however, this method allows the athlete to self-select the intensity, which could result in a reduced thermal, cardiovascular, and relative work-load [7]. One of the most popular methods of HA is the isothermal approach, which involves continuously adjusting exercise intensity to maintain an internal body temperature of 38.50 C [4]. One limitation to this method of HA is the variability in cardiovascular and workload responses between individuals. One of the largest limitations of this method is based on the overload principle of training and the fact that athletes often reach internal body temperatures well above this threshold [11]. A HA protocol that involves greater internal body temperatures may produce greater physiological and perceptual adaptations and there is a need to investigate such protocol. Since HAz in milder climates may not induce the needed adaptations for hot weather competitions, HAz and HA may be prescribed together to optimize practicality and ef- ciency while improving physiological bene ts associated with either alone, but little is known about combined protocols, especially with a more intense HA protocol. A previous study that investigated combining two forms of HA did not nd evidence that this method was any more effective than a traditional HA protocol [12]. However, the length of the second, more intense portion of HA was only implemented for 3 days. This, in addition to the altogether low levels of hyperthermia that was achieved in this protocol, leaves speculation as to whether a longer and/or more intense protocol would have resulted in similar results. Time and practicality of implementation are often limiting factors when selecting the appropriate heat mitigation strategy in sport and military settings. A unique protocol that involves 5 days of HA following HAz could offer all of the physiological and perceptual bene ts observed in 7–14 days of HA with less time required in an arti- cial laboratory, making the idea of HA implementation more appealing to

often limiting factors when selecting the appropriate heat mitigation strategy in sport and military settings. A unique protocol that involves 5 days of HA following HAz could offer all of the physiological and perceptual bene ts observed in 7–14 days of HA with less time required in an arti- cial laboratory, making the idea of HA implementation more appealing to athletes and coaches. Additionally, HAz is often already utilized in these settings and the addition of a short-term HA protocol could provide additional bene ts that could lead to enhanced performance and safety. Upon successful implementation of a short-term HA protocol to supplement HAz, sport and military personnel may be more likely to adopt this heat mitigation strategy. Therefore, the aim of this study was to assess the effectiveness of HAz followed by short-term HA on physiological and perceptual variables during steady-state exercise in

Int. J. Environ. Res. Public Health2021,18, 4366 3 of 12 the heat. We hypothesize that physiological and perceptual adaptations would be observed following HAz in an aerobically trained population, and that additional adaptations would follow a novel short-term HA protocol. Successful completion of this protocol and con rmation of these hypotheses would provide coaches, sports medicine professionals, and military personnel with an effective, practical training approach when preparing athletes and war ghters for exercise in hot environments. 2. Materials and Methods Twenty-five male endurance athletes were included in this study (age,36 12 y ; height, 178.81 6.39 cm ; body mass, 73.03 8.97 kg; and VO 2peak57.48 7.03 mL kg 1 min 1 ). These participants were recruited from the local running and cycling community. This study was approved by the University of Connecticut institutional review board and all participants provided written informed consent. A within-participant longitudinal study design was utilized (Figure). Figure 1.Study timeline. Baseline, post-heat acclimatization, and post-heat acclimation. To assess physiological and perceptual adaptations, participants completed 60 min of steady state exercise (59% 2% vVO 2peak) in an arti cial environmental laboratory (ambient temperature (T amb) 35.11 0.62 C, relative humidity (RH) 47.61% 0.38%, wet bulb globe temperature (WBGT) 29.53 0.63 C, wind speed, 4.02 0.12 mph) at three timepoints: baseline, post-HAz, and post-HAz+HA. All trials were performed on a motorized treadmill (T150; COSMED, Traunstein, Germany). These environmental conditions were chosen to re ect red ag conditions for physical activity [2]. The number of days between baseline and post-HAz were recorded (baseline and post-HAz,109 9 days ). It was assumed that participants were unacclimatized at baseline, as all participants resided in the northeastern states of the United States and the environmental conditions were higher following the baseline trial (Figure) than prior to it. Throughout the trials, physiological (HR, rectal temperature [Trec], and T sk) and perceptual (rating of perceived exertion (RPE), thermal sensation (TS), thirst, and fatigue) measures were recorded every ve minutes. HR was measured with a chest strap (H10 ® , Polar Electro™, Kempele, Finland) and participants were instructed to insert a rectal probe 10

trial (Figure) than prior to it. Throughout the trials, physiological (HR, rectal temperature [Trec], and T sk) and perceptual (rating of perceived exertion (RPE), thermal sensation (TS), thirst, and fatigue) measures were recorded every ve minutes. HR was measured with a chest strap (H10 ® , Polar Electro™, Kempele, Finland) and participants were instructed to insert a rectal probe 10 cm passed the anal sphincter for Trecto be recorded (MP160; BIOPAC Systems Inc., Goleta, CA, USA). T skwas measured on four sites (iButton; iButton Link LLC., Whitewater, WI, USA), including the thigh, chest, upper arm, and calf, and mean T skwas calculated [13]. SR was calculated by taking the difference in nude body mass measurements assessed before and immediately post exercise, accounting for urinary losses. Sweat electrolyte concentration (sodium (Na + ), potassium (K + ), and chloride (Cl )) was also assessed via the whole-body wash-down technique [14]. Participants were instructed to arrive to the

Int. J. Environ. Res. Public Health2021,18, 4366 4 of 12 laboratory euhydrated and this was con rmed with urine indices (urine speci c gravity, 1.010 0.008; and urine color, 2 0) [15]. No uid was provided throughout the 60 min of exercise. Figure 2. (a) Average maximum monthly ambient temperature in New England by climate division for April–May (baseline; unacclimatized) in 2019 and (b) average maximum monthly ambient temperature in New England by climate division for June–August (heat acclimatization) in 2019. Due to the longitudinal nature of this study, VO 2peakand vVO 2peakchanges were assessed to ensure that there were no changes in aerobic tness that could in uence the physiological variables observed in the trials. VO 2peakand vVO 2peakwas assessed prior to baseline and post-HAz. Participants were asked to don a HR monitor (H10 ® , Polar Electro™, Kempele, Finland) and complete a self-selected 5-min warm-up. Following warm-up, participants completed a graded maximal exercise test on a treadmill (T150; COSMED, Traunstein, Germany) at 2% grade to volitional exhaustion (TrueOne 2400, ParvoMedics, Sandy, UT, USA). Speci cally, the speed on the treadmill increased by 1.0 or 0.5 milesper hour (mph) at the end of each 2-min stage until volitional exhaustion. The relative volume of oxygen recorded during the nal completed stage was reported as the VO 2peakand the velocity at this stage was reported as the vVO 2peak. Following baseline trials, participants completed and recorded self-directed summer training between baseline and post-HAz. Participants utilized their own training devices (Garmin,n= 21 (Forerunner ® Fenix ® Vivoactive ® Garmin™Ltd., Olathe, KS, USA); Polar H10 and Polar Beat application,n= 3 (H10 ® , Polar Electro™, Kempele, Finland)) [16].

Int. J. Environ. Res. Public Health2021,18, 4366 5 of 12 In addition to these devices, 3 participants also utilized cycling computers to track their cycling training (Wahoo ELEMNT Bolt,n= 1 (ELEMNT Bolt, Wahoo Fitness ® , Atlanta, GA, USA), Garmin Edge,n= 1 (Edge ® , Garmin Ltd., Olathe, KS, USA), Bryton Rider 15,n= 1 (Rider 15 ® , Bryton™ Inc., Taipei City, Taiwan)). No training instruction was given during this period. Meteorological data from training sessions that were performed outside (with the exception of swimming) were extracted from the nearest available automated surface observing station (ASOS), within a mean distance of 16 11 km from the location of training. The location of training was determined by the GPS device and the latitude/longitude of that training session location was utilized to determine the closest weather station. Daytime WBGTs (7 a.m.–7 p.m.) were modeled using a Heat Stress Advisor software package (version 2005; Zunis Foundation, Tulsa, OK, USA; Coyle 2000) [17], which is designed to work with weather station data; nighttime WBGTs were computed using the Liljegren model with solar radiation set to zero [18]. Total distance, average HR, session duration, T amb, %RH, heat index, and WBGT were reported (Table). Indoor training was excluded from this analysis. Table 1. Self-directed summer training (HAz) and environmental data. Data are describing averages for each training session. Data are reported as mean standard deviation (M SD). Exercise Type (# of Sessions) Distance (km) Heart Rate (bpm) Duration (min) Heat Index ( C) WBGT ( C) Time of Day (hh:mm) Outdoor Running (n= 1692) 10.28 8.43 140 15 56.38 72.66 29.89 2.42 22.31 4.23 12:14 4:42 Outdoor Cycling (n= 364) 32.74 26.21 128 16 91.67 69.27 30.17 2.41 23.68 3.96 13:12 3:52 Multi-Sport (n= 18) 27.88 15.43 125 6 90.71 31.78 31.32 1.51 22.03 6.20 11:11 4:33 Hiking (n= 19) 8.50 8.95 94 18 161.58 170.58 30.77 4.61 19.39 6.84 11:50 3:30 Following post-HAz, participants completed a ve-day HA protocol in an arti cial en- vironmental laboratory (T amb,38.67 1.03 C ; %RH, 51.34 2.42%; WBGT,33.82 1.20 C ; wind speed, 0 0 mph). These

27.88 15.43 125 6 90.71 31.78 31.32 1.51 22.03 6.20 11:11 4:33 Hiking (n= 19) 8.50 8.95 94 18 161.58 170.58 30.77 4.61 19.39 6.84 11:50 3:30 Following post-HAz, participants completed a ve-day HA protocol in an arti cial en- vironmental laboratory (T amb,38.67 1.03 C ; %RH, 51.34 2.42%; WBGT,33.82 1.20 C ; wind speed, 0 0 mph). These environmental conditions were chosen to maximize the internal temperature response. Five HA sessions were completed within eight days and the number of days between each HA session and tests were recorded (post-HAz and HA #1 , 4 2 days; HA #1 and HA #2 , 1 1 day; HA #2 and HA #3 , 2 1 days; HA #3 and HA #4 , 2 1 days; HA #4 and HA #5 , 1 1 days; total number of HA days, 6 1 days; HA #5 and post-HA, 3 1 days). The HA sessions involved exercise to induce hyperthermia for 60 min and is termed hyperthermic zone HA (HZHA). Hyperthermia was de ned as temperatures between 38.50 and 39.75 C. In general, the exercise sessions began with a higher intensity of exercise (70% vVO 2peak) and the intensity was adjusted throughout the session to allow the participant to experience hyperthermia for 60 min. Total Trecand Trec above 38.50 C integral area under the curve (AUC) was calculated for each HA session (Table). The sample size calculation was based on the second part of this two-part study in which participants were assigned to groups following HA. The calculation was performed in G*Power (version 3.1, Dusseldorf, Germany) and is based on the variability of internal body temperature between two groups from a heat training intervention in a previous study examining the effectiveness of heat exposures every ve days following HA [19]. For a two-sided test with 0.05 alpha level and desired power level of 0.8 the estimated sample size would be 24 participants. Repeated measure ANOVAs were utilized to determine differences in physiological and perceptual outcomes between testing time points. For all analyses, in the presence of a signi cant Mauchly's test of

heat exposures every ve days following HA [19]. For a two-sided test with 0.05 alpha level and desired power level of 0.8 the estimated sample size would be 24 participants. Repeated measure ANOVAs were utilized to determine differences in physiological and perceptual outcomes between testing time points. For all analyses, in the presence of a signi cant Mauchly's test of sphericity, greenhouse–Geisser correction was used. Pairwise differences were assessed post-hoc using LSD. Cohen's d effect sizes (ES) were calculated to quantify the magnitude of pairwise differences. ES was interpreted according to the following thresholds: < 0.2 = trivial, 0.2–0.6 = small, 0.7–1.1 = moderate, 1.2–2.0 = large, and > 2.0 = very large [20]. Statistical signi cance

This study evaluates the effects of heat acclimatization and acclimation on endurance athletes' physiological responses.