Daniel Snape

The Tokyo 2020 Olympic Games were due to be the hottest on record, with hot (>30 °C) and high-humidity environmental conditions (70-90% relative humidity). Olympic Distance Triathlon comprises a 1.5 km swim, a 40 km bike ride, and a 10 km run performed successively. Prolonged exercise in the heat produces greater physiological and perceptual strain compared to exercising in temperate environmental conditions at the same intensity. Athletes competing in middle- and long-distance events are at a higher risk of reductions in exercise performance and incidence of heat illness compared to short distance events in the heat. Heat acclimation (HA) is the most effective countermeasure to protect athlete health and improve performance. Research on applied and effective HA strategies which confer the necessary heat adaptation responses while still conforming to the ecological training needs of highly trained athletes is limited. The first study (Chapter 4) of this thesis established the reliability of novel blood biomarkers and classical physiological and perceptual markers of heat stress during a new cycling heat stress test (HST) which fixes the intensity relative to body mass. The test offers a reliable assessment tool to evaluate the effectiveness of HA protocols employed. Chapter 5 evaluated whether 5-days isothermic HA was an effective preparation strategy for the Tokyo 2020 Olympic Games in endurance trained athletes. Reduced cardiovascular and perceptual strain, plasma volume expansion and an increase in sweat loss were evident. However, there was no change in body temperature or surrogate biomarkers of physiological stress. Chapter 6 investigated the effectiveness of a novel and ecologically valid 8-day HA strategy which combined modes of HA; isothermic HA and post-temperate exercise hot water immersion (HWI). There was reduced thermal, cardiovascular, and perceptual strain after 4-days HA, with no further improvement at day 8 of HA. Diminished sympathetic activity and fluid-regulatory stress were evident after 8-days HA. However, no change in performance was demonstrated during a 20-km cycling time trial in the heat. This thesis provides practitioners and coaches within British Triathlon Federation a reliable assessment tool of heat dissipation capacity in Tokyo specific environmental conditions. 5-days isothermic HA induced partial HA, affording athletes a time-efficient approach for rapid adaptation. However, a combined HA protocol of isothermic HA and HWI over 4 and 8-days induces superior thermoregulatory, perceptual, and biochemical adaptations. Hence, is the recommended approach for elite triathletes prior to competition at the Tokyo 2020 Games.

Purpose: Establish the short-term reliability and acute responsiveness of biomarkers of physiological stress to exercise in the heat. As such, informing their prospective application in research and field settings. Method: Fourteen male endurance trained cyclists/triathletes completed two heat stress tests (HST), separated by 5-7 days. HST’s involved 45-minutes fixed-intensity cycling (2.5W.kg-1) under hot-humid conditions (32oC and 70% relative humidity). Venous blood was drawn pre- and immediately post-HST for the concentration of normetanephrine (NMET), metanephrine (MET), kidney-injurymolecule1(KIM-1),neutrophilgelatinase-associatedlipocalin(NGAL),serumosmolality (Sosmo) and copeptin. Results: No biomarker displayed systematic trial order bias (p ≤ 0.05). The majority of biomarkers had acceptable within-participant variation (CV range: 0.9-14.3%). Copeptin had the lowest short-term variation at rest (CV = 0.9%) and post-HST (CV = 1.2%). However, greater variation was evident in biomarkers MET and KIM-1 at rest (CV = 28.6 & 43.2%) and post-HST (CV = 29.9 & 29.6%), respectively. NMET exhibited very large increases (trial 1 = Δ1048 ± 461; trial 2 = Δ 1067±408)inresponsetoexertionalheatstress(p<0.0001,d=2.8;p<0.0001,d=3.8).Incontrast, KIM-1 demonstrated trivial changes (trial 1 = Δ -3 ± 21; trial 2 = Δ 2 ± 17) in response to exercise in theheat (p=0.53,d=0.1;p=0.60,d=0.1).Conclusion:Eachbiomarker,exceptMETandKIM-1had acceptable reliability at rest and following exercise. In addition, biomarkers NMET, copeptin and NGAL demonstrated large increases in response to exercise in the heat. Thus, these markers can provide accurate and sensitive measurement for wide-spread application in laboratory and field research.

Abstract

Introduction Heat adaptation is protective against heat illness however its role in heat syncope, due to reflex mechanisms, has not been conclusively established. The aim of this study was to evaluate if heat acclimation (HA) was protective against heat syncope and to ascertain underlying physiological mechanisms. Method 20 (15 males, 5 females) endurance trained cyclists were randomised to either 8 days of mixed active and passive HA (HEAT) or temperate exercise (CONTROL). Prior to, and following, the interventions participants underwent a head up tilt (HUT) with graded lower body negative pressure (LBNP) continued until presyncope with measurement of cardiovascular parameters. Heat stress testing was performed to determine physiological and perceptual measures of HA. Results There was a significant increase in orthostatic tolerance (OT), as measured by HUT/LBNP, in the HEAT group (pre-intervention; 28 ± 9 mins, post-intervention; 40 ± 7 mins) compared to CONTROL (pre-intervention; 30 ± 8 mins, post-intervention; 33 ± 5 mins) (p = 0.0116). Heat acclimation resulted in a significantly reduced peak and mean rectal and skin temperature (p < 0.0141), peak heat rate (p < 0.0033), thermal comfort (p < 0.0411) and rating of perceived exertion (p < 0.0251). There was a significantly increased plasma volume (PV) in the HEAT group in comparison to CONTROL (p = 0.0293). Discussion Heat adaptation causes improvements in OT and is likely to be beneficial in patients with heat exacerbated reflex syncope. Heat acclimation mediated PV expansion is the likely predominant physiological mechanism underlying improved OT.

A mixed‐method heat acclimation (HA) protocol may optimise performance by supporting the training taper while promoting thermal adaptation; however, the impact on cardiovascular and fluid‐regulatory adjustments to protect health is unknown. Therefore, we examined the effects of a mixed‐method heat protocol on physiological responses, including cardiovascular and fluid‐regulatory strain with exercise‐heat stress, and self‐paced performance in the heat. Twenty (15 males, five females) triathletes were randomised to 8 days of HA (HOT), or exercise in thermoneutral conditions (TEMP). A heat stress test (HST) comprising 45 min of cycling in a climatic chamber (32°C, 70% relative humidity) was performed on days 1, 5 (HOT only) and 8. Before and after the intervention, a cycling time trial was conducted in the same climatic conditions (days 0 and 10). Venous blood samples were analysed at rest and post‐HST (days 1 and 8 only) for the catecholamine product normetanephrine and the vasopressin surrogate copeptin. Following 7 days of HA (days 1 vs. 8) resting rectal temperature was significantly lower in the HOT compared to the TEMP group (−0.32 ± 0.36°C, P = 0.002). Normetanephrine was 24.3% lower after 7 days of HA (P = 0.012), and copeptin was 53.4% lower at the post‐HST time point (HOT vs. TEMP, P = 0.012). However, HA had no effect (0.3%, P = 0.984) on self‐paced performance in the heat. Mixed‐method HA elicited a progressive reduction in cardiovascular strain and a net reduction in fluid‐regulatory strain without improving self‐paced performance in the heat.

Introduction Recruitment and training is vital to maintaining the size, deployability and effectiveness of armed forces, but was threatened early in the COVID-19 pandemic. Reports suggested asymptomatic seroconversion driving SARS-CoV-2 transmission in young adults. Potential association between lower vitamin D status and increased infection risk was also highlighted. We aimed to prospectively determine seroconversion and test the hypothesis that this would vary with vitamin D supplementation in representative populations. Methods Two cohorts were recruited from Yorkshire, Northern England. Infantry recruits received daily oral vitamin D (1000 IU for four weeks, followed by 400 IU for the remaining 22 weeks of training) in institutional countermeasures to facilitate ongoing training/co-habitation. Controls were recruited from an un-supplemented University population, subject to social distancing and household restrictions. Venous blood samples (baseline and Week 16) were assayed for vitamin D and anti-SARS-CoV-2 spike glycoprotein antibodies, with additional serology (weeks 4, 9, 12) by dried blood spot. Impact of supplementation was analysed on an intention-to-treat basis in volunteers completing testing at all timepoints and remaining unvaccinated against SARS-CoV-2. Variation in seroconversion with vitamin D change was explored across, and modelled within, each population. Results In the military (n=333) and University (n=222) cohorts, seroconversion rates were 44.4% vs 25.7% (P=0.003). At week 16, military recruits showed higher vitamin D (60.5 ± 19.5 mmol.L-1 vs. 53.5 ± 22.4 mmol.L-1, p < 0.001), despite <50% supplementation adherence. A statistically significant (p=0.005) effect of negative change in vitamin D (%) on seroconversion in recruits (OR of 0.991 and 95% CI of 0.984-0.997) was not evidenced in the University cohort. Conclusion Among unvaccinated populations, SARS-CoV-2 infection of infantry recruits was not reduced by institutional countermeasures, versus civilians subject to national restrictions. Vitamin D supplementation improved serum levels, but implementation did not have a clinically meaningful impact on seroconversion during military training.

• There is a growing need to develop measures for assessing performance and health status under heat stress, as heat-related health risks increase with global warming. • The ACSM recommends that a tailored heat tolerance assessment (HTA), based upon participants’ fitness levels, should be applied for individual’s affected by heat related illness. • Aerobically-trained athletes show features of heat adaptation at baseline and, in our experience, a standardised HTA to provide robust heat stress and challenge fitter individuals is lacking. • We designed a progressive HTA to be performed in uncompensable conditions, in order to minimise differences attributable to variation in body size and increase comparability.

This study investigated how exercise and heat stress may affect the neurobiomarkers Glial fibrillary acid protein (GFAP) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). Both are assayed in plasma by a semiquantitative cartridge-based system (Abbott iSTAT Alinity®) that is FDA-approved to ‘rule in’ mild Traumatic Brain Injury (mTBI) following head trauma. We hypothesised that healthy volunteers would show unchanging levels across exercise bouts differing by mode, duration, environmental conditions and heat acclimation (HA) status. We further investigated the frequency with which the manufacturer’s decision thresholds for suspecting more severe brain injury might be breached by neurobiomarker response to exercise-heat stress. Twenty endurance trained volunteers (five females, 15 males; age 30 ± 7 years, VO2max 56 ± 10 ml.kg.min-1) were sampled for blood before and after cycle ergometry in a heated chamber (45 min at 2.0 to 2.5 W.kg-1 in 32°C), at un-adapted baseline and again following eight days of HA or control activity (combined exercise-hot water immersion regimen, n=10 vs. matched temperate exercise, n=10). Separately, 50 unacclimatised runners (nine females, 39 males; age 31 ± 5 years) gave blood at rested baseline and after running the Brighton marathon 2022 (finishing time 3 h 59 min ± 49 min; peak ambient temperature 11 °C). In each study, body core temperature (Tc) was recorded (rectal thermistor vs BodyCap® telemetry pill), change in body mass (BM) was measured and blood collected and centrifuged within 30 min post-exposure. A single accredited laboratory received frozen samples for analysis of thawed plasma by both benchtop ELISA (BENCH) and iSTAT Alinity (CARTRIDGE). Cycling in the heat resulted in significant (P<0.05) overall ΔBM (–1.41 ± 0.39 kg) and ΔTc (+1.5 ± 0.4 °C). No change in neurobiomarkers was observed by BENCH or CARTRIDGE, neither did HA status influence levels (P=0.94) despite reduced Tc and increased sweat losses. CARTRIDGE decision thresholds for GFAP (30 pg.mL-1) and UCHL1 (360 30 pg.mL-1) were not exceeded. In the marathon, significant ΔBM (-1.39 ± 1.72 kg) and ΔTc (+1.8 ± 0.6 °C) were observed. Versus baseline, GFAP did not vary, whereas UCH-L1 increased for both BENCH (64.9 [39.1, 578.1] vs 91.0 [39.1, 570.0] pg.mL-1, P=0.0018) and CARTRIDGE (200 [200, 200] vs 448 [302.8, 756.0 pg.mL-1, P<0.0001). The CARTRIDGE decision threshold was not breached for GFAP, but was exceeded for UCHL1 in 32/50 runners (highest value 2261 pg.mL-1, reportable range 200 to 3200 pg.mL-1). In summary, neurobiomarkers used in mTBI assessment were unaffected by moderate intensity and duration cycling in the heat, with HA status having no discernible impact. However, prolonged running in cool weather conditions elevated UCHL1 above the decision threshold used in mTBI assessment. These results suggest caution in interpreting elevated UCHL1 after prolonged exercise.

Background and Objectives: Neurobiomarkers measured in peripheral blood can supplement management strategies following traumatic brain injury (TBI). Dual-assay of glial fibrillary acid protein (GFAP) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) is FDA-approved to inform a decision threshold approach (GFAP >30 μg.L-1 and/or UCHL1 >360 μg.L-1) for post-TBI neuroimaging. As physical activity and thermal strain often accompany TBI-prone activities, we investigated whether each molecule’s quantification - and, by extension, clinical decisions - could be influenced by exercise-heat stress. Methods: In healthy volunteers monitored continuously for body core temperature (Tc), we used the i-STAT Alinity to assess plasma GFAP and UCHL1 responses to exercise in the laboratory (four female, eighteen male trained participants, cycling for 45 min in 32 °C) and field (three female and 22 male recreational marathon runners, finishing time 231± 34 min, peak ambient temperature 11 °C). Results: Respective ΔTc overall were 1.42 ± 0.37 °C and 1.87 [1.53, 2.31] °C. With laboratory exercise, GFAP and UCHL1 did not exceed the manufacturer’s decision threshold. Across the marathon, GFAP was stable, whereas UCH-L1 more than doubled (200 [200, 200] vs 462 [310, 782] μg.L-1, P<0.0001), breaching the decision threshold for neuroimaging in 18/25 runners. Discussion: Confounding from more severe exercise-heat stress should be considered when interpreting near-care assay of UCHL1 for TBI management.

Background Detecting antibody responses following infection with SARS-CoV-2 is necessary for sero-epidemiological studies and assessing the role of specific antibodies in disease, but serum or plasma sampling is not always viable due to logistical challenges. Dried blood spot sampling (DBS) is a cheaper, simpler alternative and samples can be self-collected and returned by post, reducing risk for SARS-CoV-2 exposure from direct patient contact. The value of large-scale DBS sampling for the assessment of serological responses to SARS-CoV-2 has not been assessed in depth and provides a model for examining the logistics of using this approach to other infectious diseases. The ability to measure specific antigens is attractive for remote outbreak situations where testing may be limited or for patients who require sampling after remote consultation. Methods We compared the performance of SARS-CoV-2 anti-spike and anti-nucleocapsid antibody detection from DBS samples with matched serum collected by venepuncture in a large population of asymptomatic young adults (N = 1070) living and working in congregate settings (military recruits, N = 625); university students, N = 445). We also compared the effect of self-sampling (ssDBS) with investigator-collected samples (labDBS) on assay performance, and the quantitative measurement of total IgA, IgG and IgM between DBS eluates and serum. Results Baseline seropositivity for anti-spike IgGAM antibody was significantly higher among university students than military recruits. Strong correlations were observed between matched DBS and serum samples in both university students and recruits for the anti-spike IgGAM assay. Minimal differences were found in results by ssDBS and labDBS and serum by Bland Altman and Cohen kappa analyses. LabDBS achieved 82.0% sensitivity and 98.2% specificity and ssDBS samples 86.1% sensitivity and 96.7% specificity for detecting anti-spike IgGAM antibodies relative to serum samples. For anti-SARS-CoV-2 nucleocapsid IgG there was qualitatively 100% agreement between serum and DBS samples and weak correlation in ratio measurements. Strong correlations were observed between serum and DBS-derived total IgG, IgA, and IgM. Conclusions This is the largest validation of DBS against paired serum for SARS-CoV-2 specific antibody measurement and we have shown that DBS retains performance from prior smaller studies. There were no significant differences regarding DBS collection methods, suggesting that self-collected samples are a viable sampling collection method. These data offer confidence that DBS can be employed more widely as an alternative to classical serology.

The purpose of this study was to assess the reliability of blood biomarkers that can signify exercise-induced heat stress in hot conditions. Fourteen males completed two heat stress tests separated by 5-7 days. Venous blood was drawn pre- and post- heat stress for the concentration of normetanephrine, metanephrine, serum osmolality, copeptin, kidney-injury molecule 1 and neutrophil gelatinase-associated lipocalin. No biomarker, except copeptin, displayed systematic trial order bias (p ≥ 0.05). Normetanephrine, copeptin and neutrophil gelatinase-associated lipocalin presented acceptable reliability (CV range: 0.9-14.3%), while greater variability was present in metanephrine, osmolality and kidney-injury molecule 1 (CV range: 28.6-43.2%). Normetanephrine exhibited the largest increase (p <0.001) in response to heat stress (trial 1 = 1048 ± 461 pmol. L-1; trial 2 = 1067 ± 408 pmol. L-1), whilst kidney-injury molecule 1 presented trivial changes (trial 1 = -4 ± 20 ng. L-1; trial 2 = 2 ± 16 ng. L-1, p >0.05). Normetanephrine, copeptin and neutrophil gelatinase-associated lipocalin demonstrated good reliability and sensitivity to an acute bout of heat stress. These biomarkers may be suitable for application in laboratory and field research to understand the efficacy of interventions that can attenuate the risk of thermal injury whilst exercising in the heat.

Heat adaption through acclimatisation or acclimation improves cardiovascular stability by maintaining cardiac output due to compensatory increases in stroke volume. The main aim of this study was to assess whether 2D transthoracic echocardiography (TTE) could be used to confirm differences in resting echocardiographic parameters, before and after active heat acclimation (HA). Thirteen male endurance trained cyclists underwent a resting blinded TTE before and after randomisation to either 5 consecutive daily exertional heat exposures of controlled hyperthermia at 32◦C with 70% relative humidity (RH) (HOT) or 5-days of exercise in temperate (21◦C with 36% RH) environmental conditions (TEMP). Measures of HA included heart rate, gastrointestinal temperature, skin temperature, sweat loss, total non-urinary fluid loss (TNUFL), plasma volume and participant’s ratings of perceived exertion (RPE). Following HA, the HOT group demonstrated increased sweat loss (p = 0.01) and TNUFL (p = 0.01) in comparison to the TEMP group with a significantly decreased RPE (p = 0.01). On TTE, post exposure, there was a significant comparative increase in the HOT group in left ventricular end diastolic volume (p = 0.029), SV (p = 0.009), left atrial volume (p = 0.005), inferior vena cava diameter (p = 0.041), and a significant difference in mean peak diastolic mitral annular velocity (e’) (p = 0.044).Cardiovascular adaptations to HA appear to be predominantly mediated by improvements in increased preload and ventricular compliance. TTE is a useful tool to demonstrate and quantify cardiac HA.

ORAL PRESENTATION Purpose: To evaluate and compare the acute group and individual endogenous hormone responses to three resistance exercise workouts and two load carriage workouts. Understanding hormone responses specific to military settings may enhance training optimisation in the future. Methods: Eighteen resistance exercise-trained male civilians (age: 19 to 38 y, mean 24.9 y) completed five experimental workouts and a control condition in a randomised order; three resistance exercise workouts (cluster, hypertrophy, endurance), two load carriage (90-min constant pace [CO-LC] or 6 × 8-min intervals [IT-LC], both carrying a rucksack with 25% body mass) workouts, and a control condition (60-minutes of rest). Venous blood was drawn before, immediately post workout, and 30-min and 24-h post workout. Testosterone (T), free testosterone (fT), growth hormone (GH), dehydroepiandrosterone sulphate (DHEAS), androstenedione (ANST), and insulin-like growth factor 1 (IGF-1) concentrations were subsequently analysed via liquid chromatography-mass spectrometry. Group responses were evaluated using two-way ANOVA’s with Tukey’s post-hoc analysis. The standardised standard deviations of the pre-post change scores (SDIR) were calculated to identify the presence of individual responses (Hopkins, 2015). Results: Group responses were observed despite large individual variations. T concentrations increased in response to hypertrophy (p < 0.001) and CO-LC (p = 0.02) workouts; fT concentrations increased in response to CO-LC (p < 0.001) and decreased following the hypertrophy (p = 0.035) workout; ANST concentrations increased in response to CO-LC (p = 0.007); GH increased following hypertrophy (p = 0.004), endurance (p = 0.04), IT-LC (p < 0.001), and CO-LC (p < 0.001) workouts; IGF-1 increased in response to the endurance (p = 0.018) workout. The SDIR effect size thresholds of extremely large (T, GH, DHEAS) and very large (T, GH, DHEAS, ANST, IGF-1) were achieved, representing an individualised pattern of hormone production in response to the different resistance exercise and load carriage workouts. Conclusions: The presence of within-workout variations in the hormonal responses to all workout types suggests that the mean group responses may not reflect the response experienced by all participants, and that the hormonal anabolic response differs between individuals. The group responses and within-workout variation suggest that a wide panel of endogenous hormones should be measured to provide clarity on the anabolic response to different types of training stimuli at the individual level. Military Impact: The anabolic response to training type differs between individuals. This research provides support to further investigate whether training interventions based upon an individualised anabolic hormone response to acute exercise lead to improved physical outcomes. Funding for this research was provided by Dstl MOD. References: Hopkins W. J Appl Physiol, 118:1444–1446,2015; doi:10.1152/japplphysiol.00098.2015

Introduction Heat adaptation is protective against heat illness; however, its role in heat syncope, due to reflex mechanisms, has not been conclusively established. The aim of this study was to evaluate if heat acclimation (HA) was protective against heat syncope and to ascertain underlying physiological mechanisms. Methods Twenty (15 males, 5 females) endurance-trained athletes were randomized to either 8 d of mixed active and passive HA (HEAT) or climatically temperate exercise (CONTROL). Before, and after, the interventions participants underwent a head up tilt (HUT) with graded lower body negative pressure (LBNP), in a thermal chamber (32.0 ± 0.3°C), continued until presyncope with measurement of cardiovascular parameters. Heat stress tests (HST) were performed to determine physiological and perceptual measures of HA. Results There was a significant increase in orthostatic tolerance (OT), as measured by HUT/LBNP, in the HEAT group (preintervention; 28 ± 9 min, postintervention; 40 ± 7 min) compared with CONTROL (preintervention; 30 ± 8 mins, postintervention; 33 ± 5 min) (P = 0.01). Heat acclimation resulted in a significantly reduced peak and mean rectal and skin temperature (P < 0.01), peak heat rate (P < 0.003), thermal comfort (P < 0.04), and rating of perceived exertion (P < 0.02) during HST. There was a significantly increased plasma volume (PV) in the HEAT group in comparison to CONTROL (P = 0.03). Conclusions Heat acclimation causes improvements in OT and is likely to be beneficial in patients with heat exacerbated reflex syncope. Heat acclimation–mediated PV expansion is a potential physiological mechanism underlying improved OT.