2 3 Running Title: vs. twice-daily heat acclimation · 18 (TDHA) in comparison to once-daily heat acclimation (ODHA) and work matched once- or twice-daily 19 temperate exercise (ODTEMP,

and Rehabilitation (CHPER),

1 Title: Once- and twice-daily heat acclimation confer similar heat adaptations, inflammatory responses

2 and exercise tolerance improvements.

3 Running Title: Once- vs. twice-daily heat acclimation

4 Authors:

5 1A.G.B Willmott, 1M. Hayes, 1,2C.A James, 1J. Dekerle, 1,3O.R Gibson and 1N.S Maxwell

6 Address for Authors:

7 1Environmental Extremes Laboratory, University of Brighton, Eastbourne, UK, 2Institut Sukan Negara

8 (National Sports Institute), National Sports Complex, Kuala Lumpur, Malaysia, 3Centre for Human

9 Performance, Exercise Brunel University London, Uxbridge, UK

10 Details for the

11 Ashley Willmott – [email protected]

12 Word Count:

13 Abstract Word Count:

14 Tables: 5

15 Figures: 3

Corresponding Author:

16 Abstract

17 This experiment aimed to investigate the efficacy of twice-daily, non-consecutive heat acclimation

18 (TDHA) in comparison to once-daily heat acclimation (ODHA) and work matched once- or twice-daily

19 temperate exercise (ODTEMP, TDTEMP) for inducing heat adaptations, improved exercise tolerance,

20 and cytokine (immune) responses. Forty males, matched biophysically and for aerobic capacity, were

21 assigned to ODHA, TDHA, ODTEMP or TDTEMP. Participants completed a cycling graded exercise

22 test, heat acclimation state test and a time to task failure (TTTF) at 80% peak power output in temperate

23 (TTTFTEMP: 22°C/40% RH) and hot conditions (TTTFHOT: 38°C/20% RH), before and after 10-sessions

24 (60-min of cycling at ~2W.kg-1) in 45°C/20% RH (ODHA and TDHA) or 22°C/40% RH (ODTEMP

25 or TDTEMP). Plasma IL-6, TNF-α and cortisol were measured pre- and post-sessions 1, 5 and 10.

26 ODHA and TDHA induced equivalent heat adaptations (P<0.05) (resting rectal temperature [-

27 0.28±0.22, -0.28±0.19°C], heart rate [-10±3, -10±4 b.min-1] and plasma volume expansion [+10.1±5.6,

28 +8.5±3.1%]) and improved heat acclimation state (sweat setpoint [-0.22±0.18, -0.22±0.14°C] and gain

29 [+0.14±0.10, +0.15±0.07g.sec-1.°C-1]). TTTFHOT increased (P<0.001) following ODHA (+25±4%) and

30 TDHA (+24±10%), but not ODTEMP (+5±14%) or TDTEMP (+5±17%). TTTFTEMP did not improve

31 (P>0.05) following ODHA (+14±4%), TDHA (14±8%), ODTEMP (9±10%) or TDTEMP (8±13%).

32 Acute (P<0.05) but no chronic (P>0.05) increases were observed in IL-6, TNF-α or cortisol during

33 ODHA and TDHA, or ODTEMP and TDTEMP. Once- and twice-daily heat acclimation conferred

34 similar magnitudes of heat adaptation and exercise tolerance improvements, without differentially

35 altering immune function, thus non-consecutive TDHA provides an effective, logistically flexible

36 method of HA, benefitting individuals preparing for exercise-heat stress.

37 New and Noteworthy

38 • Greater heat adaptations and enhanced exercise performance in the heat were induced by 10-

39 sessions of consecutive once-daily and non-consecutive twice-daily heat acclimation, compared

40 with equivalent temperate exercise, without adverse inflammatory or stress responses.

41 • No difference in the magnitude of adaptation and enhanced exercise performance were observed

42 between either non-consecutive twice-daily, or consecutive once-daily heat acclimation when

43 protocols were matched for volume and intensity.

44 • Non-consecutive twice-daily heat acclimation provides an alternate method to consecutive once-

45 daily heat acclimation to induce heat adaptation without requiring consecutive day training.

46 Glossary of terms 47 Blood lactate concentration – [La]b

48 Body surface area – BSA

49 Body mass index – BMI

50 Change – ∆

51 Cycling graded exercise test – GXT

52 Gross mechanical efficiency – GME

53 Heart rate – HR

54 Heat acclimation – HA

55 Heat acclimation state test – HAST

56 Interleuken-6 – IL-6

57 Lactate threshold – LT

58 Long- term heat acclimation – LTHA

59 Medium- term heat acclimation – MTHA

60 Metabolic heat production – Ḣprod

61 Once-daily heat acclimation – ODHA

62 Onset of blood lactate accumulation – OBLA

63 Peak oxygen uptake – V̇O2

64 Peak power output – PPO

65 Plasma volume – PV

66 Rating of perceived exertion – RPE

67 Rectal temperature – Tre

68 Relative humidity – RH

69 Respiratory exchange ratio – RER

70 Short-term heat acclimation – STHA

71 Sodium concentration – [Na+]

72 Thermal comfort – TC

73 Thermal sensation – TSS

74 Time to task failure – TTTF

75 Time to task failure in heat stress – TTTFHOT

76 Time to task failure in temperate conditions – TTTFTEMP

77 Tumour necrosis factor-alpha – TNF-α

78 Twice-daily heat acclimation – TDHA

79 Urine colour – Ucol

80 Urine osmolality – Uosm

81 Urine specific gravity – Usg

82 Ventilation – V̇E

83 Volume of oxygen uptake – V̇O2

84 Whole-body sweat loss – WBSL

85

86 Introduction

87 Heat acclimation (HA) is an important preparation strategy preceding exercise-heat stress (64, 70) to

88 alleviate physiological strain (61), attenuate heat related illness (HRI) (94), improve thermal perception

89 (33) and exercise tolerance in hot (56), and possibly temperate conditions (50). A variety of HA

90 strategies currently exist, predominantly differentiated by exercise-heat stress volume, and/or intensity

91 (18, 80). In this regard, HA may be applied within sporting and occupational settings (e.g. military),

92 with current recommendations advocating the use of repeated, consecutive once-daily exertional heat

93 exposures for 60-100-min, utilising an isothermic protocol (70). In spite of multiple manipulations of

94 volume/intensity, the optimal frequency for HA remains largely unknown (83). Current

95 recommendations for once-daily exposures are implied more readily than non-consecutive [e.g. 10-

96 sessions in 21-days (32)] and twice-daily exposures [e.g. 100-min vs. 2x50-min (49)], due to the

97 consistency of potentiating stimuli for adaptation e.g. daily elevations in rectal [Tre] and skin

98 temperature alongside profuse sweating, which are required to evoke a multitude of physiological and

99 perceptual adaptations (74). From a practical perspective, implementing consecutive-day protocols is

100 challenging given access to hot-humid conditions is not ubiquitous, and the need for daily exposures is

101 likely to interrupt sport/occupational-specific training, competition tapering and, or travel/recovery

102 schedules. Medium- (MTHA: 10-14-days) and long-term (LTHA: >14-days) protocols which maximise

103 adaptations exacerbate these challenges, a factor which may provide some explanation as to why, in

104 spite of clear recommendations from the scientific community, only ~15% of athletes undertook HA

105 prior to competition in heat stress (66).

106 We have previously shown that four HA sessions i.e. a short-term HA (STHA) intervention (89),

107 administered over two consecutive days (i.e. twice-daily HA [TDHA]), demonstrated comparable

108 adaptations to four consecutive once-daily HA (ODHA) sessions. However the magnitudes of

109 adaptation using STHA are typically smaller than MTHA/LTHA interventions, thus the need to

110 examine the efficacy of a twice-daily approach over longer periods exists. Furthermore given challenges

111 associated with consecutive day interventions, completing TDHA intermittently (e.g. over non-

112 consecutive days), over MTHA/LTHA timescales, may be desirable given an improved ability to

113 integrate HA into complex training and travel schedules, potentially reducing disruption. For example,

114 by administering the same number of HA sessions (i.e. the same dose) non-consectutively, athletes may

115 be afforded recovery days during HA or have the ability to perform specific training on non-HA days.

116 Whilst hypothetically beneficial, investigations are needed to assess the efficacy of this strategy,

117 particularly given different markers of heat adaptation have differing timecourses for induction (67)

118 and the associations between adaptation and performance enhancement are not ubiquitously reported.

119 Previous research findings are equivocal, with sub-optimal adaptations reported during non-consecutive

120 versus daily HA (32), attributable to heat decay (88) and insufficient physiological stimulus (4).

121 Consequently, refining non-consecutive protocols so that the timescale, protocol and dose are in line

122 with best practice recommendations i.e. using an isothermic model of ~10-sessions over 10-14-days

123 (70) and thus, ensuring twice-daily methods implement appropriate potentiating stimuli, may ameliorate

124 current limitations and provide an alternative strategy for practitioners who pursue HA benefits but

125 prioritise training quality and recovery schedules.

126 Whilst acute exercise-heat stress is unlikely to impair immune function (84, 85), few studies have

127 investigated immunological biomarkers during HA despite the potential for immunological

128 perturbations to culminate in exacerbated inflammatory (e.g. interleukin-6 [IL-6] and tumour necrosis

129 factor-alpha [TNF-α]) and stress responses (e.g. cortisol) (14, 92), potentially increasing HRI

130 susceptibility (48) and diminishing the application and efficacy of HA (34, 69). Investigation of

131 inflammatory responses to once-daily isothermic HA reported few negative findings (14), however the

132 immune response to our proposed twice-daily model of matched volume (dose), but altered frequency,

133 remains unknown and maladaptation may be a concern. Therefore, investigation is required given the

134 repeated exercise-induced hyperthermia, coupled with shorter recovery time during the ‘heat days’ of

135 TDHA that may result in an overload of physiological strain, inducing residual stress between sessions

136 (72).

137 This study investigated the efficacy of short- (i.e. 5-sessions) and medium-term (i.e. 10-sessions) HA,

138 using non-consecutive TDHA and consecutive ODHA protocols, and compared these to temperate

139 exercise groups (i.e. once-daily: ODTEMP and non-consecutive twice-daily: TDTEMP) as frequency

140 and duration matched exercise controls. Secondly, this study investigated exercise tolerance through

141 the determinants of aerobic performance, and subsequent performance in both hot and temperate

142 conditions between interventions. Finally, this study also investigated the inflammatory and stress

143 responses during interventions to determine whether a compromised immune function was an artefact

144 of the twice-daily protocol. It was hypothesised that as the dose of HA was the same, TDHA would

145 induce the same physiological and ergogenic benefits as ODHA, with both TDHA and ODHA superior

146 to ODTEMP and TDTEMP. Given the alteration in frequency of the HA dose, it was hypothesised that

147 the reduced duration between TDHA sessions would lead to undesirable inflammatory/stress responses

148 in comparison to ODHA.

149 Methods

150 Participants and ethical approval

151 Forty moderately-trained [performance level 3 (62)] males provided informed consent to participate in

152 the experiment, which was approved by the Univeristy of Brighton Institution’s Research Ethics and

153 Governance Committee and conducted in accordance with Declaration of Helsinki (2013). Participants

154 were matched for biophysical characteristics and aerobic capacity and assigned to; consecutive ODHA,

155 non-consecutive TDHA, consecutive ODTEMP or non-consecutive TDTEMP. No differences in

156 participant characteristics were observed (P>0.05 [Table 1]),

157 ***Add Table 1 near here***

158 Experimental design

159 Prior to group allocation, participants completed four tests comprising; cycling graded exercise test

160 (GXT), heat acclimation state test (HAST) and time to task failure test in hot (TTTFHOT) and temperate

161 conditions (TTTFTEMP), in a semi-randomised order, 48-hr apart with the GXT completed first.

162 Interventions consisted of, 60-min exercise sessions performed in hot (45°C, 20% RH) or temperate

163 conditions (22°C, 40% RH) over a 12-day period. Post-tests were repeated in the same order 48-hr apart

164 (Figure 1). This study was completed during November-February, with trials occurring at the same time

165 of day to minimise the effect of circadian variation on exercise tolerance (21) and thermoregulation

166 (86). Participants avoided alcohol and caffeine 12-hr before experimentation, arrived in a euhydrated

167 state (73) and replicated food intake the day of the each exercise trial (2).

168 ***Add Figure 1 near here***

169 Determinants of aerobic performance - Graded exercise test (GXT)

170 Height (Detecto Scale Company, USA) and body mass (Adam Equipment Inc., USA) were measured,

171 enabling the estimation of body surface area (BSA) (5). Skinfold thickness was measured (Harpenden,

172 Baty International, UK) across four sites (22) to estimate body fat (%) (76). The GXT was completed

173 on an electronically-braked stationary ergometer (SRM High performance model, Germany) within

174 temperate conditions (22°C, 40% RH). Power output was initially set at 80 W and increased by 24 W

175 every stage (3-min), with cadence kept at 80 rev.min-1. Capillary blood lactate concentration ([La]b)

176 was sampled within the final 30-s of each stage and analysed immediately (2300 Plus, YSI, USA).

177 Breath-by-breath metabolic gas data were continuously collected (Metalyzer 3B, Cortex, Germany).

178 Lactate threshold (LT) was determined by an increase (>1 mmol.L-1) in [La]b above resting level (15)

179 and the test was terminated when the onset of blood lactate accumulation (OBLA) occurred (>4 mmol.L-

180 1) (93). Gross mechanical efficiency (GME) was calculated from steady-state oxygen consumption and

181 respiratory exchange ratio (RER <1.0) values collected during the final 30-s of each stage of the LT test

182 (25). Following 15-min rest, participants performed a second test with an initial power output 48 W

183 below OBLA that was increased by 20 W.min-1 until volitional exhaustion (39). Peak oxygen uptake

184 (V̇O2peak) and power output (PPO) were determined as the highest average V̇O2 and power output during

185 the final 30-s of each stage. Following 15-min rest, participants were familiarized to the TTTF at 80%

186 of their PPO.

187 Aerobic performance - Time to task failure (TTTF)

188 TTTFTEMP (22°C, 40% RH) and TTTFHOT (38°C, 40% RH) were completed at 80% of PPO (51) on a

189 modified cycle ergometer (SRM crankset and wireless PowerControl meter on a Monark 874E,

190 Sweden). Following a standardised warm-up (2-min seated rest, 5-min at 90% of LT, 3-min rest and

191 then 3-min of unloaded pedalling at 80 rev.min-1), power output was increased to 80% PPO. HR, Tre

192 and metabolic gas data were collected every minute and RPE was recorded at task failure (i.e. when

193 cadence failed <77 rev.min-1 for >3-s following a warning). Power output, HR and time were obscured

194 with only cadence displayed.

195 Heat acclimation state test (HAST)

196 HASTs were completed in hot-dry conditions (45°C, 15% RH) within an environmental chamber (TISS,

197 UK) on a cycle ergometer (Monark 620, Sweden). HASTs simulated Havenith and Middendorp (1986)

198 protocol, but prescribed exercise intensities at given rates of Ḣprod relative to body mass (3.0, 4.5 and

199 6.0 W.kg-1) (91). Heat acclimation state was identified via sweat setpoint and sweat gain measures (37).

200 Metabolic energy expenditure was estimated from known values of V̇O2 and RER below LT during the

201 GXT (58). Ḣprod was subsequently calculated and associated exercise intensities prescribed (Cramer and

202 Jay 2014) during the HAST, which were re-calculated post-intervention.

203 Heat acclimation and temperate exercise protocols

204 Participants completed ten 60-min exercise sessions over 12-days. Once-a-day groups (ODHA,

205 ODTEMP) exercised on days 1-5 and 8-12 at 08:00-hr, whereas twice-daily groups (TDHA, TDTEMP)

206 exercised twice on days 1, 3, 8 and 10 at 08:00-hr and 16:00-hr, and then once on days 5 and 12 at

207 08:00-hr (Figure 1). Exercise commenced at 2.3 W.kg-1 (~65% V̇O2peak) for 15-min at 80 rev.min-1, in

208 line with recommended guidelines to rapidly attain the desired change in core temperature (31, 41).

209 Power output was subsequently altered depending on changes in Tre (∆Tre) and perceived effort (55), to

210 target a Tre of ≥38.5°C for the remainder of the session (80) (see Table 2 for actual training data). To

211 amplify ∆Tre, upper-body sauna suits (Everlast, London, UK) (90) were worn during the initial 15-min

212 of exercise. This method has been applied prior to HA (53) to increase physiological strain without

213 increasing exercise intensity or volume (19, 90). Physiological and perceptual measures were recorded

214 at rest and every 5-min during exercise for all 10 sessions. During sessions 1, 5 and 10, fluid ingestion

215 was prohibited for accurate estimation of sweat loss. Participants were permitted to drink ab libitum

216 during the remaining sessions (55). Euhydration was determined on arrival to each session by collection

217 of mid flow urine; colour <3 (Ucol), osmolality <700 mOsmol.kg-1 (Uosm) (Osmocheck, Vitech Scientific

218 Ltd., Japan) and specific gravity <1.020 (Usg) (hand refractometer, Atago, Japan) (73). HR was

219 manually recorded (Polar Electro, Oy, Finland) and Tre was continuously monitored using a thermistor

220 probe (Henleys Medical Supplies, UK) self-inserted 10 cm past the anal sphincter. Whole-body sweat

221 loss (WBSL) was estimated for each session from towel dried nude body mass differences pre- to post-

222 exercise. Sweat samples (~2 mL) were collected in a Tegaderm+Pad (3MTM, USA) placed on the

223 midpoint of the trapezius before being analysed for sodium concentration ([Na+]) using a Sweat-ChekTM

224 (Eli Tech Group, Wescor Inc., USA) for sessions 1, 5 and 10.

225 Phlebotomy and biochemistry

226 Following 10-min of seated rest immediately before and after sessions 1, 5 and 10, fingertip capillary

227 blood (~200 µL) was sampled for haemoglobin (HemoCue, Ltd., Sweden) and haematocrit (Hawksley

228 and Sons Ltd., England) to estimate ΔPV (20). A 10 mL venepuncture sample was also collected from

229 the antecubital fossa, transferred into two 5 mL tubes (EDTA Sarstedt, Akteingesellscaft and Co,

230 Germany), centrifuged (Eppendorf 5702 R Centrifuge, UK) for 10-min at 5000 rev.min-1, and then

231 plasma stored at -86°C. Upon analysis, commercially available ELISA kits were used to measure IL-6

232 and TNF-α (Ready Set Go!®, eBioscience, Affymetrix Inc., USA) and cortisol (Sigma-Aldrich, USA)

233 in duplicate and corrected for ∆PV.

234 Perceptual measures

235 RPE (6) from 6 (No exertion) to 20 (Maximal Exertion), thermal sensation scale [TSS (82) from 0 (Very

236 Very Cold), 4 (Neutral) to 8 (Very Very Hot)] and thermal comfort [TC (95) from 0 (Very Comfortable)

237 to 5 (Very Uncomfortable)], were collected during exercise sessions every 5-min following

238 familiarisation.

239 Data and statistical analyses

240 All data are reported as mean ± SD, with statistical significance set at P<0.05. Data were assessed and

241 conformed to normality and sphericity prior to further statistical analysis. Within-group differences for

242 pre-intervention data sets were analysed using a one-way ANOVA. To assess intervention efficacy,

243 physiological, performance and perceptual data were analysed using a three-way mixed design ANOVA

244 (Time*Condition*Frequency), for time (pre- to post-intervention), condition (HA and TEMP) and

245 frequency (once- and twice-daily exercise). Following a significant F-value, follow up Bonferroni-

246 corrected post-hoc comparisons were used. Predefined analytical limits to highlight meaningful heat

247 adaptations were; ∆Tre >0.20°C, ∆HR >5 b.min-1 ∆WBSL >200 mL, ∆PV >5% and >1 in perceptual

248 scales (RPE, TSS and TC) (92). Typical error of measurement (TEM) were used to determine

249 meaningful differences for sweat setpoint (0.21°C), sweat gain (0.09 g.sec-1.°C-1), TTTF test (15%),

250 V̇O2peak test (4.8%), IL-6 (2 pg.mL-1), TNF-α (1 pg.mL-1) and cortisol (57 nmol.L-1). Isotime data (i.e.

251 task failure time-point pre-intervention compared to the corresponding time-point post-intervention)

252 was also analysed.

253 Results

254 Heat adaptations

255 During both ODHA and TDHA interventions, resting Tre, resting HR, and sweat [Na+] were reduced,

256 while WBSL and PV were increased within session 5 (STHA) and 10 (MTHA) (P<0.05) compared to

257 session 1 (Table 2). The highest recorded perceptual measures (i.e. peak RPE, TSS and TC) were also

258 lower (P<0.05) from session 1-5 (STHA), and 1-10 (MTHA) during ODHA and TDHA. These

259 physiological and perceptual adaptations were greater following session 10 (MTHA) compared to

260 session 5 (STHA) (P<0.05). Adaptations did not differ between HA groups (all P>0.05), but larger

261 magnitudes in adaptations were observed compared to both TEMP interventions (P<0.05) (Table 2).

262 There were no differences (P>0.05) between groups for exercise time, intensity or work completed

263 during the HA or TEMP sessions. However, as expected physiological strain (i.e. time >38.5°C and

264 ∆Tre) was larger (P<0.05) during HA compared to TEMP (Table 3). Exercise time and work completed

265 during exercise sessions were greater (P<0.05) between session 1-10 (MTHA) and session 1-5 (STHA)

266 for each group (Table 3).

267 Post-intervention HASTs demonstrated reductions in sweat setpoint, HRpeak and TCpeak, and

268 improvements in sweat gain and WBSL (P<0.05) for ODHA and TDHA groups, with greater

269 improvements compared to TEMP (P<0.05), yet no differences were found between HA protocols

270 (P>0.05) (Table 2).

271 ***Add Table 2 and 3 near here***

272 Exercise tolerance

273 Determinants of aerobic performance - GXT

274 A main effect was found for power output at LT and V̇O2peak (P<0.05), with a greater (P<0.05)

275 improvement following HA (ODHA and TDHA), compared to TEMP (ODTEMP and TDTEMP; Table

276 4). No Time*Condition*Frequency interaction (P>0.05) was found for any GXT data. No

277 improvements (P>0.05) were found in PPO or GME.

278 Aerobic performance - TTTF

279 Pre-intervention TTTFHOT was shorter (all P<0.001) compared to TTTFTEMP for all groups, with no

280 between-group differences (P>0.05).

281 TTTFHOT improved (P<0.001) following ODHA and TDHA, but not ODTEMP or TDTEMP (P>0.05),

282 whereas TTTFTEMP did not improve (P>0.05) following any intervention (Table 4). Following TDHA

283 and ODHA only, Tre and HR were lower at isotime (P<0.05) during TTTFHOT and TTTFTEMP (Table 5).

284 ***Add Table 4 and 5, and Figure 2 near here***

285 Biomarkers

286 Increased plasma [IL-6], [TNF-α] and [cortisol] (P<0.05) were observed from pre- to post-session 1, 5

287 and 10 during both HA and TEMP protocols (Figure 3). Inflammatory and stress responses were greater

288 for HA compared to TEMP with larger mean: ∆IL-6 values following session 1, 5 and 10 (P<0.001);

289 ∆TNF-α following session 1 and 10, but not 5 when comparing HA to ODTEMP only (P<0.05); and

290 ∆cortisol following session 5 for ODHA vs. TEMP, and following session 5 and 10 for TDHA vs.

291 TEMP (Figure 3). No differences in inflammatory or stress responses were observed between the HA

292 protocols at any time point (P<0.05). Interestingly, there was no evidence of chronic effects over the

293 course of HA or TEMP (P>0.05), however there was a trend (P<0.10) for the ∆IL-6 and ∆cortisol to be

294 lower and ∆TNF-α to be higher for session 10 when compared to the other sessions for ODHA and

295 TDHA only.

296 ***Add Figure 3 near here***

297 Discussion

298 In agreement with our hypothesis, ODHA and TDHA induced comparable heat adaptations to one

299 another, thus demonstrating an improved heat acclimation state compared to ODTEMP and TDTEMP.

300 Improvements in power at LT and V̇O2peak were found following HA, in addition to both ODHA and

301 TDHA enhancing performance (TTTF) in hot, but not temperate conditions, an improvement that was

302 not observed by either TEMP group. Inflammatory responses increased acutely following single

303 sessions in all groups, with larger responses during HA vs TEMP. However, contrary to out hypothesis,

304 no difference was observed between ODHA and TDHA groups. These data highlight that non-

305 consecutive TDHA presents no difference to ODHA, inducing similar heat adaptation and

306 improvements in exercise tolerance during heat stress, without compromising immune status. These

307 findings suggest the dose of HA (e.g. matched weekly exposure and intensity) is most important for the

308 mechanisms which underpin adaptation, as opposed to the structure of HA (e.g. frequency [once- or

309 twice-daily] and timing [morning or afternoon]).

310 Heat adaptations

311 HA efficacy was confirmed by the acquisition of key physiological heat adaptations including

312 reductions in resting Tre (-0.3°C) and HR (-10 b.min-1), [Na+] retention (-14 to -27 mmol.L-1) and,

313 increased WBSL (+398 to +533 mL) and PV expansion (+8.5 to +10.1) (Table 2). Proportional

314 improvements were also observed following just 5-sessions (i.e. STHA). Reductions in RPE (-2) and

315 TSS (-0.7 to -0.9), and an improved TC (-1), also demonstrate positive perceptual improvements

316 following 10-sessions of both ODHA and TDHA. Collectively, these adaptations are in line with a

317 recent meta-analysis on HA (83) and, whilst direct comparisons across studies are difficult due to

318 differences in HA exercise protocols, MTHA studies (i.e. once-daily) do report equivalent magnitudes

319 of adaptation to the present study [e.g. resting Tre: -0.17°C, and HR: -5 b.min-1, [Na+] retention: -

320 22mmol.L-1, WBSL: +29% and PV expansion: +4.3% (83)]. Moreover, ODHA and TDHA induced

321 adaptation superior to our predefined analytical limits (∆Tre >0.20°C, ∆HR >5 b.min-1, ∆WBSL >200

322 mL, ∆PV >5% and >1 in perceptual scales [RPE, TSS and TC] (92)) highlighting meaningful heat

323 adaptations, a critical factor when assessing intervention strategies.

324 Both HA strategies improved heat acclimation state, as indicated by a lower sweat setpoint (-0.22°C)

325 and a larger sweat gain (+0.14 to +0.15 g.sec-1.°C-1) during the post-intervention HAST (Table 2).Whilst

326 no reductions in ∆Tre, Trepeak, RPEpeak or TSSpeak occurred following HA, this can be explained by the re-

327 prescription of exercise intensities, thus, controlling for Ḣprod post-intervention and providing

328 confidence in our adaptations. The unchanged Tre but larger WBSL (both +35%) shows

329 thermosensitivity is enhanced via increased sweat gain for ODHA (+48%) and TDHA (+49%). Though

330 theese changes are superior to the meta-analysis findings (+25% (83)) and TEM (0.09 g.sec-1.°C-1 (91))

331 the authors accept that an oesophageal core temperature and real-time local sweat rate measurements

332 would offer superior assessment of these data given a more rapid response in comparison to our rectal

333 thermistor (81). Parallel reductions in resting Tre and sweat setpoint, following ODHA (-0.28 and -

334 0.22°C) and TDHA (-0.28 and -0.22°C), respectively, agree with meta-analysis findings (-0.28 (83))

335 and are larger than the TEM (0.21°C (92)). MTHA (e.g. 10-sessions) induced greater magnitudes of

336 physiological and perceptual heat adaptation compared to STHA (e.g. 5-sessions [Table 2 and 3]).

337 Though not in agreement with all experimental data (27, 28), these findings agree with consensus

338 recommendations that longer-term HA (e.g. ≥10-days) is preferable to induce greater physiological heat

339 adaptations (67, 70) achieved in this study through the maintained physiological strain imposed using

340 isothermic prescription (80). These data provide supporting evidence that medium- to long-term HA

341 could be prescribed immediately before, or potentially several weeks before major athletic competition

342 or military deployment in heat stress (18) to induce greater initial adaptations, as opposed to solely

343 implementing STHA during the final training microcycle. This notion, alongside the decay of these

344 aforementioned adatations (18), should be experimentally examined as this strategy would allow

345 alternate approaches (e.g. intermittent ‘top up’ exposures in the days preceding exposure) to be

346 implementated to maintain the enhanced heat acclimation state (8).

347 Seminal work by Lind and Bass (49) demonstrated the benefits of continuous, once-daily HA (i.e. 100-

348 min sessions), as opposed to longer and shorter intermittent times (e.g. twice-daily, 2x50-min), which

349 contributed to duration recommendations for optimal heat adaptations (70). Our data indicate no

350 advantage but more importantly, no disadvantage of non-consecutive TDHA over consecutive ODHA,

351 agreeing with our previous STHA investigation (89). Further to this, as outlined above, these

352 observations are true even when the session duration is 60 min (this study), as opposed to 90 – 100 min

353 which has been previously described as preferable (70). Our novel findings are in contrast to others

354 which have not demonstrated efficacy of TDHA (32), but may be explained by a) the use of an

355 isothermic model, b) the matching of exercise-heat dose (e.g. duration, intensity and total number of

356 exposures) to induce equivalent heat adaptations and improved exercise tolerance, and/or c) more

357 significant heat strain i.e. maximising time spent at the targeted Tre. This non-consecutive twice-daily

358 structure is likely to be appealing to coaches and practitioners with upcoming competitions in

359 challenging, hot conditions (e.g. Tokyo 2020 Olympic and Paralympic Games) for whom scheduling

360 HA around sport-specific training, competition tapering, rest and travel is challenging. This study is the

361 first to demonstrate equivalent heat adaptations following both TDHA and ODHA, with greater

362 adaptations for longer interventions (i.e. 5- vs. 10-days) suggesting the dose of HA (i.e. attaining key

363 physiological responses to a greater extent) is the primary factor that underpins adaptation.

364 Exercise tolerance

365 Determinants of aerobic performance

366 Our study provides a holistic overview of the changes in exercise tolerance following non-consecutive

367 TDHA, in comparison to consecutive ODHA and matched TEMP interventions. V̇O2peak improved

368 following HA (ODHA: +4.6%; TDHA: +3.7%), with this change greater than TEMP changes

369 (ODTEMP: +2.6%; TDTEMP: +1.4%). This is likely due to hypervolemia following HA and potential

370 increments in cardiac output (50) however it must be acknowledged that participants were not elite

371 athletes whom as a cohort may be less responsive to this mechanism (59). Nonetheless, previous studies

372 report ergogenic benefits of HA on V̇O2peak and PPO in temperate conditions (23, 24, 50, 71, 75, 79)

373 whilst others present no changes (43, 44, 55). Power at LT also improved significantly following HA,

374 in agreement with previous findings (9, 10, 42, 45, 50, 55, 68, 71) however improvements following

375 ODHA (+7±10 W) and TDHA (+7±8 W) were of. A lower magnitude than those reported in well-

376 trained cyclists in 13°C (+12-15 W (50)) and 22°C (+16 W [(55)] and +15 W [(71)]). Furthermore,

377 GME did not change following interventions, in agreement with previous LTHA (43). Whilst the

378 ergogenic benefits of HA remain disputed between research groups, potentially as a result of insufficient

379 potentiating stimuli or inter-individual differences (13), our data are the first to demonstrate that

380 implementing a non-consecutive twice-daily intervention does not induce differential ergogenic effects

381 to that of a matched dose once-daily protocol, for the determinants of aerobic performance (e.g. V̇O2peak

382 and power at LT) in temperate conditions.

383 Aerobic performance - Time to task failure Determinants of aerobic performance

384 TTTFHOT improved following ODHA (+25%) and TDHA (+24%), but not ODTEMP and TDTEMP

385 (both +5%), agreeing with previous reports following MTHA (+67% [(56)], +17% [(57)] and +24%

386 [(17)]), which appear to exceed STHA (+14% [(26)] and +7% [(11)]) likely due to greater physiological

387 adaptation. Evidence for TTTFHOT improvements likely reflecting the magnitude HA adaptations (e.g.

388 PV expansion improving cardiac output (50, 56), leading to increased V̇O2peak and power at LT, resulting

389 in a lessened physiological strain (67), is indicated by a lower mean Tre (-0.26°C) and HR (-8 b.min-1)

390 at isotime (Table 5). Consequently, non-consecutive TDHA appears equally effective as ODHA for

391 improving aerobic performance (e.g. extending exercise tolerance time) in sub-elite athletes within the

392 severe-intensity domain under heat stress. It is likely adaptations contributed to the improved TTTFTEMP

393 following ODHA and TDHA (both +14%). However, these data describe that HA (irrespective of once-

394 or twice-daily frequency) provided only moderate ergogenic benefits for performance in temperate

395 conditions, opposing significant time trial improvements in 13°C (50) and 22°C (71) but agreeing with

396 recent studies which suggest these physiological constructs are not limitng (43, 44, 55). Nonetheless,

397 this is the first study to collectively assess TTTF in both hot and temperate conditions which whilst

398 demonstrating some inter-individual differences (Figure 3), describes ODHA and TDHA as providing

399 ergogenic benefits for enhanced performance in hot conditions with data in temperate conditions

400 encouraging, albeit not unequivocal (54, 60).

401 Inflammatory and stress responses

402 Agreeing with previous literature, larger ∆IL-6 and ∆TNF-α were observed during HA compared to

403 TEMP (Figure 2) (3, 34, 35, 46, 47, 63, 77). The larger responses observed for ∆cortisol for session 5

404 and 10, but not 1 (1, 7, 12, 36, 40, 63, 77) during HA are a response to increased physiological strain

405 due to the heat stress for the same absolute exercise intensity (e.g. higher Tre, ∆Tre and HR) (77). Our

406 changes in IL-6 (+55%), TNF-α (+45%) and cortisol (+34%) during HA were comparable in ODHA

407 and TDHA, and are less than, or comparable to, responses published elsewhere (IL-6: +20-2000%,

408 TNF-α: +15-65% and cortisol: +20-70%) (1, 3, 7, 14, 34, 35, 40, 46, 63, 77). Our findings also agree

409 with reported transient ∆IL-6 during MTHA (14, 34) alongside induced heat adaptations (71) and no

410 evidence of chronic inflammatory effects or signs of exaggerated ∆TNF-α (e.g. possible endotoxemia)

411 (34). The absence of augmented ∆cortisol as HA progresses, conforms to previous literature describing

412 the sensitivity of this biomarker to various stressors (1, 14, 26, 78, 87). In summary, our data indicates

413 no chronic inflammatory effects or stress responses during ODHA and, for the first time during non-

414 consecutive TDHA, which is likely due to the equivalent acquisition in physiological heat adaptation.

415 These novel data provide confidence that our TDHA protocol did not induce unexpected inflammatory

416 or stress responses which could compromise immune status in subsequent heat exposures to any greater

417 extent than ODHA. This further strengthens the argument for TDHA when ODHA is impractical.

418 Application

419 The similarity of the responses to non-consecutive TDHA and ODHA, may be of particular interest to

420 sporting and occupational organisations that require heat adaptations to lessen the physiological strain

421 and HRI risk, and improve exercise performance in heat stress. Non-consecutive TDHA provides an

422 alternate and flexible strategy, providing the potential to half the number of interrupted training days,

423 thus maximising an individual’s time to complete specific (e.g. non-heat) training or rest/recover

424 without compromising the magnitude of adaptation. Logistically, the non-consecutive TDHA is

425 appealing given the cost and time associated with athletes or workers travelling to specialist heat

426 training facilities in cool climates, may be reduced if multiple heat sessions can be completed on one

427 day. The transient nature of heat adaptations requires STHA during crucial preparation periods, where

428 training is predominantly sport-specific with volume often adjusted to optimise recovery, thus resulting

429 in training that opposes targetted physiological adaptations. It is unsurprising therefore, that repeated

430 steady-state exercise during consecutive day HA, do not appear to be widely embraced by competitive

431 athletes (65). Prescribing TDHA and specifically afternoon sessions, may also increase HA efficiency

432 as time spent at the desired isothermic Tre of >38.5°C was extended during afternoon compared to

433 morning sessions (+14 vs. +6 min), yet ∆Tre were lower (+1.3°C vs. +1.6°C), thus requiring less exercise

434 time to reach target temperatures due to circadian rhythm and higher resting Tre. Ultimately, shorter

435 duration HA (~60 min) that provides sufficient physiological strain to evoke meaningful phenotypic

436 adaptations irrespective of daily frequency and consecutive scheduling is desirable, with non-

437 consecutive TDHA providing greater flexibility than a consecutive day protocol.

438 Limitations and future direction

439 Despite our biomarker data indicating TDHA does not induce excessive inflammatory/immune

440 responses, our mechanistic insights are limited due to the number and timing of blood sampling.

441 Collecting additional biomarker measures and across more time-points during the recovery phase (e.g.

442 1-24-hr) would provide further insight into the inflammatory responses and potential maladaptive

443 influences on the magnitude and kinetics of heat adaptation. An extension of this work would also

444 examine intracellular heat shock proteins (46) and the relevant gene transcripts (27) to elucidate the

445 impact of TDHA vs. ODHA on attaining thermotolerance (46), and potential benefits across

446 environmental stressors (29, 30). We also highlight a need to investigate the precise effect of

447 consecutive and non-consecutive TDHA in females, who experience different thermoregulatory

448 adaptation kinetics to males (52). Moreover, the effect of HA duration should be considered (e.g. 60-

449 vs. 90/100-min sessions) given an extended heat dose may impact the kinetics and magnitude of both

450 adaptation and the inflammatory responses. A paucity of data still exists to effectively characterise the

451 rate of heat decay and re-induction of HA at a physiological and molecular level, which is critical for

452 the implementation of all HA protocols including TDHA. Finally, we highlight the need for

453 investigations regarding the feasibility and appropriateness of HA and other concurrent training (e.g.

454 interval or competition specific intensity sessions) for elite athletes.

455 Conclusion

456 This is the first study to investigate the efficacy of non-consecutive twice-daily HA compared to daily

457 HA for adaptations, biomarkers and exercise tolerance. Greater heat adaptations were induced by both

458 once- and twice-daily HA protocols, compared with equivalent temperate exercise, without adverse

459 effects on inflammatory or stress responses. Exercise tolerance in heat stress was improved following

460 both HA protocols, yet no effect was found for matched-volume TEMP, nor were improvements found

461 for exercise tolerance in temperate conditions for all interventions. The concomitant increase in power

462 at LT and V̇O2peak following HA, reaffirms the erogenicity of HA on aerobic performance within heat

463 stress, although our data do not provide supportive evidence for HA to enhance aerobic performance in

464 temperate conditions.

465 Acknowledgments

466 The authors would like to thank the all the participants who volunteered for this study.

467 Conflict of interest

468 The authors confirm there are no conflict of interest.

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706 Figure Legends

707 Figure 1. Schematic design of the study. Note. HAST, TTTFHOT and TTTFTEMP performed in 708

randomised order. GXT = graded exercise test, HAST = heat acclimation state test, TTTF = time to task 709

failure in hot (HOT) or temperate (TEMP) conditions, ODHA = once daily heat acclimation, TDHA = twice 710

daily, non-consecutive day heat acclimation, ODTEMP = once daily temperate training, TDTEMP = 711 twice

daily, non-consecutive day temperate training.

712 Figure 2. Mean ± SD Mean ± SD changes in the determinants of aerobic performance and aerobic 713

performance in hot and temperate conditions. *represents a significant (P<0.05) within-group 714

difference pre- to post-session. ∂ represents a significant (P<0.05) between-group difference with (HA 715 vs.

TEMP). Shapes denote individual participants within group.

716 Figure 3. Mean ± SD changes in cortisol, TNF-α and IL-6 for session 1, 5, 10. *represents a 717

significant (P<0.05) within-group difference pre- to post-session. †represents a significant 718

(P<0.05) between-group difference with ODTEMP. ‡represents a significant (P<0.05) between- 719

group difference with TDTEMP. Shapes denote individual participants within group.

720

721 Tables

722 Table 1. Mean ± standard deviation (SD) participant characteristics.

723 Group

(n = 40)

Age

(years)

Body

mass (kg)

Height

(m)

BMI

(kg.m2)

BSA

(m2)

Sum of

skinfolds

Body fat

(%)

(n = 10)

(n = 10)

(n = 10)

(n = 10)

BMI: body mass index, BSA: body surface area, ODHA: once-daily heat acclimation, TDHA:

twice-daily heat acclimation, ODTEMP: once-daily temperate exercise and TDTEMP: twice-

daily temperate exercise.

(mm)

ODHA 23±6 77.2±10.0 1.78±0.08 24.4±2.1 1.95±0.16 34.5±7.3 14.9±2.7

TDHA 25±7 75.3±9.5 1.79±0.04 23.4±2.5 1.94±0.13 33.4±9.9 14.3±3.7

ODTEMP 22±1 77.3±8.6 1.77±0.04 25.5±3.0 1.92±0.10 35.7±6.4 15.0±1.7

TDTEMP 22±1 75.2±7.8 1.78±0.07 23.8±1.5 1.93±0.14 33.8±7.5 14.6±2.9

O

724

Table 2. Mean ± SD changes (∆) in heat adaptations over days 1-5 (short-term) and days 1-10 (medium-term) and during the heat acclimation state pre-post intervention.

Group ODHA TDHA ODTEMP TDTEMP Session 1-5 1-10 1-5 1-10 1-5 1-10 1-5 1-10

Heat adaptations

∆Rest Tre(°C) -0.18±0.27* -0.28±0.22*†‡+ -0.22±0.17*†‡ -0.28±0.19*†‡+ +0.03±0.21 -0.10±0.16 -0.04±0.17 -0.11±0.18 ∆Rest HR (b.min-1) -5±1* -10±3*+ -5±5* -10±4*+ -1±1 -2±1 +1±3 -2±6 ∆PV (%) +6.3±4.0 +10.1±5.6*+ +5.4±4.0 +8.5±3.1*+ +0.5±2.8 +1.5±3.5 +1.5±3.4 +0.7±4.1 ∆WBSL (mL) +230±207* +533±261*†‡+ +178±142*†‡ +398±97*†‡+ +83±86 +81±97 +48±68 +90±118 ∆ [Na+] (mmol.L-1) -13±13*†‡ -27±19*+†‡+ -7±6 -14±5*+ -12±12 -24±20+ -6±12 -11±13+ ∆RPEpeak -1±1 -2±1*+ -1±1 -2±1*+ -1±1 -2±1*+ 0±2 0±2 ∆TSSpeak -0.3±0.4 -0.7±0.5*+ -0.5±0.5 -0.9±0.5*+ +0.4±0.5 +0.2±0.8 +0.1±0.9 +0.1±0.7 ∆TCpeak -1±1 -1±1* 0±1 -1±1*+ 0±0 0±0 0±0 0±0

Heat acclimation state (1-10)

∆Sweat setpoint (°C) -0.22±0.18*† -0.22±0.14*‡† -0.14±0.18* -0.11±0.10* ∆Sweat gain (g.sec-1.°C-1) +0.14±0.10* +0.15±0.07* +0.05±0.07 +0.06±0.06 ∆WBSL (mL) +262±180* +278±211* +68±118 +68±112 ∆Trepeak (°C) -0.25±0.11* -0.28±0.11* -0.15±0.27 -0.08±0.25 ∆HRpeak (b.min-1) -13±9* -14±10* -4±1 -2±6 ∆RPEpeak -3±2* -3±1* -3±2* -2±2* ∆TSSpeak -0.7±0.5 -0.6±0.7 -0.4±0.9 -0.3±0.4 ∆TCpeak -1±1*‡ -1±1*‡ -1±1* 0±1 *represents a significant (P<0.05) within-group difference, †represents a significant (P<0.05) between-group difference with ODTEMP, ‡represents a significant (P<0.05) between-group difference with TDTEMP, and +represents a significant difference (P<0.05) between 1-5 and 1-10 adaptations. ODHA: once-daily heat acclimation, TDHA: twice-daily heat acclimation, ODTEMP: once-daily temperate exercise, TDTEMP: twice-daily temperate exercise, Tre: rectal temperature, HR: heart rate, PV: plasma volume, WBSL: whole-bbody sweat loss, [Na+]: sodium concentration, RPE: rating of perceived exertion, TSS: thermal sensation and TC: thermal comfort.

725

For ev ew

O

Table 3. Mean ± SD exercise data for sessions 1-5 (short-term) and 1-10 (medium-term).

Group ODHA (44.4±1.2°C, 21.1±2.4 % RH)†‡

TDHA (44.3±1.3°C, 22.2±3.9 % RH)†‡

ODTEMP (21.6±1.1°C, 40.9±4.2 % RH)

TDTEMP (21.8±1.0°C, 38.6±4.7 % RH)

Session 1-5 1-10 1-5 1-10 1-5 1-10 1-5 1-10

Time (min) 300±0 600±0*+ 300±0 600±0*+ 300±0 600±0*+ 300±0* 600±0*+

Total work (kJ) 2378±280 4838±573*+ 2338±211 4751±374*+ 2419±199 4834±405*+ 2361±254* 4778±440*+

Mean power (W.kg-1) 1.7±0.1 1.7±0.2 1.7±0.1 1.7±0.1 1.7±0.1 1.7±0.1 1.6±0.2 1.6±0.2

Mean power (% PPO) 49±4 49±3 47±5 47±5 47±5 47±5 46±6 46±6

Trepeak (°C) 38.45±0.33 38.39±0.36 38.52±0.24 38.44±0.21 38.39±0.32 38.27±0.26 38.15±0.22 38.16±0.30

∆Tre (°C) 1.29±0.21†‡∂ 1.52±0.23*†‡+∂ 1.48±0.22†‡∂ 1.68±0.28*†‡+∂ 0.70±0.17 0.69±0.18 0.78±0.17 0.90±0.19

Time >38.5°C (min) 62.8±61.2†‡∂ 118.6±118.1*†‡+∂ 50.2±30.3†‡∂ 90.9±49.5*†‡+∂ 21.3±24.5 23.5±17.0 21.5±24.7 23.5±18.8

HRpeak (b.min-1) 167±15 163±13 163±11 155±11+ 169±19 167±17 177±9 163±6+

Sweat loss (mL) 980±287 1513±504*†‡+ 1146±429†‡ 1545±375*†‡+ 655±95 736±139 616±150 733±150

∆PV (%) -7.9±4.0 -4.4±2.8+ -9.4±5.5 -4.6±3.3+ -3.2±2.6 -2.3±1.1 -2.2±1.4 -1.8±1.1

RPEpeak 15±1† 13±1*†‡+ 15±1† 14±1*†+ 17±1 16±2* 16±1 16±2

TSSpeak 6.8±0.4†‡ 6.1±0.6*+ 7.0±0.4†‡ 6.1±0.4*+ 5.8±0.5 5.9±0.7 6.2±0.7 6.1±0.5

TCpeak 4±1 2±1*+ 4±1 3±1*+ 3±0 3±1 3±0 3±0

*represents a (P<0.05) within-group difference, †represents a (P<0.05) between-group difference with ODTEMP, ‡represents a (P<0.05) between-group difference with TDTEMP, +represents a significant difference (P<0.05) between 1-5 and 1-10 adaptations and ∂represents a significant (P<0.05) between-intervention difference (e.g. HA vs. TEMP). ODHA: once-daily heat acclimation, TDHA: twice-daily heat acclimation, ODTEMP: once-daily temperate exercise, TDTEMP: twice-daily temperate exercise, Tre:

rectal temperature, HR: heart rate, PV: plasma volume, ∆: change, RPE: rating of perceived exertion, TSS: thermal sensation and TC: thermal comfort. 726

727

or R vi w l y

Determinants of Aerobic Performance

728

Table 4. Mean ± SD changes (∆) in exercise tolerance (determinants of aerobic performance and aerobic performance).

Group ODHA TDHA ODTEMP TDTEMP

Pre Post ∆ (% ∆) Pre Post ∆

(% ∆) Pre Post ∆ (% ∆) Pre Post ∆

(% ∆)

Power at LT (W) 159±20 166±26 +7±10∂

(+3.4±4.9) 163±30 170±28 +7±8∂

(+4.6±5.4) 157±21 160±23 +3±5 159±17 160±13 +1±6

(+1.9±2.8) (+1.1±4.1)

GME (%) 19.9±1.0 21.0±2.0 +1.0±2.2 20.5±1.7 20.8±1.4 +0.2±1.6 19.3±1.7 19.2±1.6 -0.1±1.5 19.7±1.9 19.7±2.0 +0.1±1.2

V̇O2peak (L.min-1) 3.76±0.46 3.95±0.52 +0.18±0.12∂

(+4.6±3.1) 3.74±0.50 3.89±0.45 +0.13±0.09∂

(+3.7±2.8) 3.73±0.43 3.83±0.45 +0.10±0.09 3.69±0.34 3.73±0.31 +0.05±0.07

(+2.6±2.5) (+1.4±2.0)

PPO (W) 291±39 304±48 +13±18 (+4.2±5.7) 296±50 308±46 +11±8

(+3.9±3.3) 288±27 291±31 +3±14 287±18 296±18 +6±11

(+1.6±4.1) (+2.3±4.1)

Aerobic Performance

TTTFTEMP (s) 519±151 588±153 +68±11 (+14±4) 553±74 631±82 +78±47

(+14±9) 510±102 553±106 +42±51 (+9±10) 532±116 579±161 +47±62

(+8±18)

TTTFHOT (s) 412±111 516±140* +104±31 (+25±4)* 450±85 558±117* +109±57

(+24±11)* 416±131 435±149 +19±58 (+5±14) 430±91 444±97 +15±77

(+5±17) *represents a significant (P<0.05) within-group difference and ∂represents a significant (P<0.05) between-intervention difference (e.g. HA vs. TEMP). ODHA: once-daily heat

acclimation, TDHA: twice-daily heat acclimation, ODTEMP: once-daily temperate exercise, TDTEMP: twice-daily temperate exercise, LT: lactate threshold, GME: gross mechanical efficiency, V̇O2peak: peak oxygen uptake, PPO: peak power output, TTTFTEMP: time to task failure in temperate condition and TTTFHOT: time to task failure in heat stress.

729

730

Re v ew

731

732

733

734

735

736

737

738

739

740

741

Table 5. Mean ± SD changes (∆) in physiological measures compared to pre-intervention time to task failure in temperate (TTTFTEMP) and hot conditions (TTTFHOT).

Group ODHA TDHA ODTEMP TDTEMP

TTTFTEMP

∆Tre (°C) ∆HR(b.min-1)

-0.21±0.12* -6±8*

-0.29±0.24* -6±4*

-0.14±0.16 +1±7

-0.14±0.28 -3±10

∆V̇O2 (L.min-1) -0.02±0.21 0.00±0.17 -0.03±0.32 -0.02±0.28 ∆RER -0.08±0.15 -0.01±0.10 -0.06±0.05 -0.07±0.08 ∆Ḣprod (W) -26±73 -28±72 +36±127 +12±170 ∆V̇E (L.min-1) -8.4±20.1 +5.2±16.0 +8.1±16.3 -6.2±21.4

TTTFHOT

∆Tre (°C) -0.26±0.26* -0.26±0.27* -0.14±0.28 -0.16±0.33 ∆HR(b.min-1) -6±5* -8±6* 0±6 -3±7 ∆V̇O2 (L.min-1) +0.01±0.29 -0.09±0.20 +0.03±0.22 -0.05±0.12 ∆RER -0.01±0.08 +0.02±0.06 -0.04±0.10 -0.07±0.08 ∆Ḣprod (W) -17±104 -11±99 +16±179 +20±209 ∆V̇E (L.min-1) -5.5±20.4 -2.3±16.0 +5.7±20.7 +1.9±16.0 *represents a significant (P<0.05) within-group difference. ODHA: once-daily heat acclimation, TDHA: twice-daily heat acclimation, ODTEMP: once-daily temperate exercise, TDTEMP: twice-daily temperate exercise, Tre: rectal temperature, HR: heart rate, V̇O2: oxygen uptake, RER: respiratory exchange ratio, Ḣprod: metabolic heat production, V̇E: ventilation, ∆: change, TTTFTEMP: time to task

failure in temperate condition and TTTFHOT: time to task failure in heat stress.

ew

Week 1 Monday Tuesday Wednesday Thursday Friday Saturday Sunday

Preliminary and GXT

(22°C, 40% R.H.)

Rest

HAST

(45°C, 20% R.H.)

Rest

TTTFHOT

(38°C, 40% R.H.)

Rest

TTTFTEMP

(22°C, 40% R.H.)

Week 2 Monday Tuesday Wednesday Thursday Friday Saturday Sunday

Morning (08:00)

ODHA, TDHA,

ODTEMP, TDTEMP

ODHA

ODTEMP

ODHA, TDHA,

ODTEMP, TDTEMP

ODHA

ODTEMP

ODHA, TDHA,

ODTEMP, TDTEMP

Rest

Rest Afternoon

(16:00)

TDHA,

TDTEMP

Rest

TDHA,

TDTEMP

Rest

Rest

Week 3 Monday Tuesday Wednesday Thursday Friday Saturday Sunday

Morning (08:00)

ODHA, TDHA,

ODTEMP, TDTEMP

ODHA

ODTEMP

ODHA, TDHA,

ODTEMP, TDTEMP

ODHA

ODTEMP

ODHA, TDHA,

ODTEMP, TDTEMP

Rest

Rest Afternoon

(16:00)

TDHA,

TDTEMP

Rest

TDHA,

TDTEMP

Rest

Rest

Week 4 Monday Tuesday Wednesday Thursday Friday Saturday Sunday

GXT (22°C, 40% R.H.)

Rest

HAST

(45°C, 20% R.H.)

Rest

TTTFHOT

(38°C, 40% R.H.)

Rest

TTTFTEMP

(22°C, 40% R.H.)

For Review

Only

Figure 2. Mean ± SD Mean ± SD changes in the determinants of aerobic performance and aerobic performance in hot and temperate conditions. *represents a significant (P<0.05) within-group difference pre- to post-session. ∂ represents a significant (P<0.05) between-group difference with (HA vs. TEMP).

Shapes denote individual participants within group.

For Review

Only

Figure 3. Mean ± SD changes in cortisol, TNF-α and IL-6 for session 1, 5, 10. *represents a significant (P<0.05) within-group difference pre- to post-session. †represents a significant (P<0.05) between-group

difference with ODTEMP. ‡represents a significant (P<0.05) between-group difference with TDTEMP. Shapes denote individual participants within group.