Menthol Mouth Rinsing Maintains Relative Power Production during Three-Minute Maximal Cycling Performance in the Heat Compared to Cold Water and Placebo Rinsing

revious menthol studies have demonstrated ergogenic effects in endurance-based activity. However, there is a need for research in sports whose physiological requirements exceed maximal aerobic capacity. This study assessed the effects of 0.1% menthol mouth-rinsing upon a modi ed three-minute maximal test in the heat (33.0 3.0 C; RH 46.0 5.0%). In a randomised crossover single blind placebo-controlled study, 11 participants completed three modi ed maximal tests, where each trial included a different mouth rinse: either menthol (MEN), cold water (WAT) or placebo (PLA). Participants were asked to rate their thermal comfort (TC), thermal sensation (TS) and rating of perceived exertion (RPE) throughout the test. Heart rate, core temperature, oxygen uptake (VO 2), ventilation (VE) and respiratory exchange ratio (RER) were monitored continuously throughout the test, alongside cycling power variables (W; W/kg). A blood lactate (BLa) level was taken pre- and post-

placebo (PLA). Participants were asked to rate their thermal comfort (TC), thermal sensation (TS) and rating of perceived exertion (RPE) throughout the test. Heart rate, core temperature, oxygen uptake (VO 2), ventilation (VE) and respiratory exchange ratio (RER) were monitored continuously throughout the test, alongside cycling power variables (W; W/kg). A blood lactate (BLa) level was taken pre- and post- test. Small to moderate effects (Cohen'sdand accompanying 90% con dence intervals) between solutions MEN, WAT and PLA were observed towards the end of the test in relation to relative power. Speci cally, from 75–105 s between solutions MEN and WAT (ES: 0.795; 90% CI: 0.204 to 1.352) and MEN and PLA (ES: 1.059; 90% CI: 0.412 to 1.666), this continued between MEN and WAT (ES: 0.729; 90% CI: 0.152 to 1.276) and MEN and PLA (ES: 0.791; 90% CI: 0.202 to 1.348) from 105–135 s. Between 135–165 s there was a moderate difference between solutions MEN and WAT (ES: 1.058; 90% CI: 0.411 to 1.665). This indicates participants produced higher relative power for longer durations with the addition of the menthol mouth rinse, compared to cold water or placebo. The use of menthol (0.1%) as a mouth rinse showed small performance bene ts for short duration high intensity exercise in the heat. Keywords:menthol; cycling; maximal exercise; heat 1. Introduction Menthol is an organic compound with the ability to impart non-thermal cooling effects on targeted receptors, across body regions. Menthol is utilised in many commercial products for its perceptual cooling effect and is nontoxic for human consumption when administered at commercial doses [1,2]. Perceptual cooling via menthol is an extension of thermal cooling for athletes performing exercise in the heat. Original thermal cooling modalities included ice slurry ingestion, cold air exposure and cold-water immersions, which typically lead to a physiological reduction in core temperature (Tcore) [3,4]. The recent combination of menthol with other thermal cooling methods has sparked interest in both research and applied settings as a novel and safe ergogenic aid, when administered in accordance with the scienti c literature [1]. It is important to acknowledge that although menthol does

and cold-water immersions, which typically lead to a physiological reduction in core temperature (Tcore) [3,4]. The recent combination of menthol with other thermal cooling methods has sparked interest in both research and applied settings as a novel and safe ergogenic aid, when administered in accordance with the scienti c literature [1]. It is important to acknowledge that although menthol does not prevent heat gain or reduce Tcore, it aids in the athletes feeling perceptually cool and being able to perform for a longer period of time [5–7] or at a Int. J. Environ. Res. Public Health2022,19, 3527.

Int. J. Environ. Res. Public Health2022,19, 3527 2 of 19 higher power output [8–10]. As long as human maximum temperature is not reached and other heat illness factors are minimised [1,11], improvements in thermal comfort have been shown to enhance performance independent of an athlete's thermal state [12,13]. There have been multiple studies that have assessed endurance exercise performance in the heat alongside menthol administration [5,14,15]. Trials that consist of exercise protocols exceeding ten minutes in duration have seen positive effects of menthol use through both topical and oral applications [1]. Topical application of menthol preceded studies on rinsing or ingestion. Gillis et al. [14], were among the rst studies to use menthol in a spray form onto the skin, using menthol concentrations of 0.01% and 0.2%, in a spray form applied to the participant's body ve minutes prior to the trial starting. Using menthol as a mouth rinse followed on from the topical spray, with similar concentrations examined. Mouth rinsing may induce a more rapid and intense experience of menthol and its cooling properties, due to decreased thickness of the stratum corneum and potential for increased stimulation of transient receptor potential melastatin 8 (TRPM8) receptors and trigeminal nerves via the oral cavity [16–18], lower concentrations may be preferred by some athletes, so as to minimize any potential irritant effects. Cold water also warrants consideration as a simple and effective rinse during exer- cise. Its temperature will also stimulate TRPM8 and trigeminal pathways, as a result of a physiological reduction in local temperature within the oral cavity. Ingestion of cool/cold beverages are preferred during exercise compared to room temperature or warm bever- ages [19–22], and are deemed to be more palatable, thus promoting further ingestion or satisfying thirst to a greater extent. The particular sensation that is imparted by `cold' is that of being of refreshed [23–25]. This has the potential to decrease the rating of perceived exertion or improve task motivation [20], along with potentially modifying sensations of air ow or nasal patency [26,27]. If suf cient uid volumes are also ingested, then a reduction in core temperature may

greater extent. The particular sensation that is imparted by `cold' is that of being of refreshed [23–25]. This has the potential to decrease the rating of perceived exertion or improve task motivation [20], along with potentially modifying sensations of air ow or nasal patency [26,27]. If suf cient uid volumes are also ingested, then a reduction in core temperature may also contribute to improvements in performance [19,20], however ice ingestion is considered a more time-ef cient means of achieving a desired reduction in core temperature, and indeed when menthol is co-ingested with a liquid, has been shown to increase its ergogenicity inversely proportional to the beverage tempera- ture [10]. Taking the above into account the inclusion of cold-water swills or ingestion in menthol trials is warranted due to their common stimulatory pathways, but different physiological outcomes. As mentioned, similar concentrations of menthol to those administered topically have been used as a mouth rinse, with 0.01% the most commonly applied to date. Studies have typically shown that the use of menthol during endurance-based activities has resulted in a smaller effect on thermal perception compared to other cooling methods such as ice slurry and cold-water immersion or application [2,17,28]. Menthol being applied as a mouth rinse targets a smaller thermally sensitive area compared to a topical spray, or other more aggressive cooling methodologies that elicit a reduction in core body temperature. However, a greater menthol concentration could potentially eliminate variability due to individuals' differing menthol sensitivity thresholds [14,29–31]. It is important to note that at higher concentrations menthol acts as an irritant, but when administered at appropriate concentrations and time intervals, irritation and desensitization are mitigated [32,33]. This indicates that the timing of menthol administration relative to, or within, an exercise bout is a pertinent consideration for practitioners and athletes. Considering current evidence, a recent consensus statement found insuf cient re- search regarding the effect of menthol on either intermittent or explosive exercise, with literature exploring the effect of menthol on short duration high-intensity bouts of exercise also scarce [1,34]. Previously, longer durations have been examined, with participants completing trials to exhaustion

is a pertinent consideration for practitioners and athletes. Considering current evidence, a recent consensus statement found insuf cient re- search regarding the effect of menthol on either intermittent or explosive exercise, with literature exploring the effect of menthol on short duration high-intensity bouts of exercise also scarce [1,34]. Previously, longer durations have been examined, with participants completing trials to exhaustion or extended time trials that rely on aerobic energy provi- sion [5,14,35]. When exercising in the heat, these durations permit a suf cient `hyperthermic dose', with Tcoresigni cantly elevated as a correlate of exercise duration. More speci cally, as exercise duration in the heat increases, so too does the cardiovascular, thermoregula- tory and ventilatory strain experienced by an athlete [36,37]. The time-course of these

Int. J. Environ. Res. Public Health2022,19, 3527 3 of 19 physiological responses presents a seemingly ideal metabolic milieu for menthol to exert its primary perceptual cooling effects and its secondary physiological effects, such as in- creasing ventilation (VE) [15]. When exercising in the heat for a short duration at near maximal intensity, the hyper-thermic potential of the exercise bout is limited, but some physiological responses akin to when athletes experience hyperthermia may still occur, due to the intensity of the exercise bout. These may include but are not limited to increases in VE, respiratory exchange ratio and carbohydrate oxidation [38,39], effectively increasing the percentage of maximal oxygen uptake (VO2max) assigned to the task. The three-minute all-out test [40,41] was designed to elicit maximal oxygen uptake, and pro le the rate of decay to critical power—capturing the asymptote of the severe intensity domain. The three-minute duration is important, as this appears to be the du- ration above (and the intensity below) which athletes demonstrate an increased aerobic contribution to energy production [38,39,42]. Using the three-minute duration also simu- lates competitive events such as the individual or team pursuit, and is mirrored in events such as 1500 m running races, where the contribution from aerobic energetic pathways are maximal (i.e., VO2max), but also require a notable energetic contribution from glycolytic sources too [38,39,42], event tactical demands notwithstanding. Given the previous work in prolonged exercise tasks and the limited evidence in short duration efforts following menthol rinsing, the three-minute test is a natural progression in the line of inquiry; as it assesses short duration and maximal effort exercise simultaneously, which have only been investigated following menthol previously to a limited extent, employing repeated sprint designs [34,43]. Based on the rationale presented above, the aim of the present study was to examine whether a single menthol mouth rinse in uenced performance of and/or thermal perception during a three-minute maximal test compared to cold water and placebo mouth rinses under hot environmental conditions. 2. Materials and Methods Ethical approval for this study was obtained from the Waikato Institute of Technology's Human Ethics Research Group (Approval

aim of the present study was to examine whether a single menthol mouth rinse in uenced performance of and/or thermal perception during a three-minute maximal test compared to cold water and placebo mouth rinses under hot environmental conditions. 2. Materials and Methods Ethical approval for this study was obtained from the Waikato Institute of Technology's Human Ethics Research Group (Approval code: WTFE13130820). Participants gave verbal and written consent upon arrival at the laboratory prior to participation and had the opportunity to ask questions about study design, intensity etc. to ensure informed consent was maintained. 2.1. Participants Eleven healthy non-heat-acclimated male (n= 6) and female (n= 5) participants (height: 171.2 11.0 cm; mass: 75 13 kg; age: 25 5 years) were included in this study. To ensure non-heat-acclimated participation, research was conducted over autumn and winter months, with no participant travelling to hot ( 28 C) climate for two months, prior to study commencement [28,31]. Inclusion criteria required participants to have general tness ability, with prior experience in exercise testing procedures and being self-reported as free from injury. Participants cycled three to seven times per week, for either dedicated exercise, competitive or cross-training purposes. All participants were familiar with the use of a WattBike™at self-reported maximal intensity. Participants were instructed to avoid non-habitual consumption of alcohol or caffeinated products 24 h prior to each testing session, as well as abstain from vigorous exercise 24 h before sessions. Participants reported to the laboratory to complete introductory measures that included, height, body mass and health questionnaires. 2.2. General Design All tests were performed on a WattBike™Pro cycle ergometer (Watt Bike Ltd., Notting- ham, UK). During the rst visit participants completed a VO2maxtest, before completing a familiarisation session and three experimental sessions in the heat chamber. Participants undertook ve visits in total.

Int. J. Environ. Res. Public Health2022,19, 3527 4 of 19 The VO2maxtest protocol was completed in temperate conditions (20 C; 30% humid- ity). Participants completed a self-selected 10 min warm up before starting the test. The same warm up was then repeated prior to experimental sessions. Participants started the incremental ramp test cycling at 100 W, this increased by 25 W every minute [44]. RPE was measured at the end of each one-minute stage by the participant pointing to a CR-10 RPE scale [45] attached to a board in front of the WattBike™. Oxygen uptake was continuously measured, breath-by-breath, using a Metalyzer CPET device (Coretex Medical, Leipzig, Germany). Maximal oxygen uptake was considered to have occurred at the point of VO2 not increasing despite an increase in VE, as per guidelines from Taylor [46] with secondary criteria of a RER > 1.10, high blood lactate levels or RPE approaching or at maximal lev- els [47]. Blood sampling for lactate analysis was not taken during the rst visit. If these criteria were not met, but participants still reached voluntary exhaustion, then the measure was deemed to be VO 2peak. During the second visit a full familiarisation session was conducted in which the par- ticipants experienced the testing conditions in an environmental heat chamber (33.0 3.0 with RH 46.0 5.0%), where the experimental tests were going to take place. This session included instructions on how to perform the three-minute all-out maximal test, as well an introduction to the thermal perception scales that were used in the testing sessions, experi- ence with potential heat stress and advice on how to recognise heat illnesses throughout the trial. WattBike™geometry measurements were also established during this session and maintained throughout the study. All participants were asked to raise questions or queries they might have had with the experimental testing and its conditions at this time, but still retained their right to withdraw at any time. Participants then performed three randomised experimental trials on visits 3–5, which were separated by at least 48 h, to permit adequate recovery. Each of the trials were performed at the same

to raise questions or queries they might have had with the experimental testing and its conditions at this time, but still retained their right to withdraw at any time. Participants then performed three randomised experimental trials on visits 3–5, which were separated by at least 48 h, to permit adequate recovery. Each of the trials were performed at the same time of the day to minimise potential circadian variation. A general experimental schematic is presented in Figure. Figure 1. Experimental overview. Circles indicate time points as indicated in Methods and/or Results sections. Mass and hydration were assessed upon entry. The dashed horizontal arrow at the top of the image indicates continuous measurement at the sampling rates mentioned in the appropriate methodological subsections, from the point of menthol mouth-rinsing (green cup). Vertical arrows at the bottom of the image indicate measurement of rating of perceived exertion, thermal comfort and thermal sensation, and blood lactate. Created with

Int. J. Environ. Res. Public Health2022,19, 3527 5 of 19 2.3. Experimental Procedure Having abstained from vigorous exercise >24 h prior to the tests, all participants were asked to ensure they were well hydrated before arriving at the laboratory. Upon arrival at the laboratory, participants were required to provide a urine sample for a urine speci c gravity test (USG) to check their hydration status. If participants USG was 1.030, they were asked to ingest 200–500 mL of water before entering the heat chamber [48]. Participants were also advised to consume a habitual diet to minimise the difference in muscle glycogen levels and respiratory exchange ratio (RER) in between visits. Participants were provided with a rectal thermistor, which was self-inserted 10 cm beyond the anal sphincter. Participants' body masses were assessed prior to entering the heat chamber and again on exit from the heat chamber to gauge sweat loss for rehydration purposes before ingesting any uid. When in the heat chamber, the rectal thermistor was connected to a data logger (SQ2010, Grant Instruments, Cambridge, UK) which recorded rectal temperature (Trec) throughout the duration of the test(s). Whilst it is acknowledged that the short testing duration was unlikely to elicit hyperthermia, in line with institutional safety requirements, internal temperature was closely monitored while in the heat chamber to ensure prevention of heat induced illnesses. Upon entering the heat chamber (33.0 3.0 C with RH 46.0 5.0%), participants were asked to make themselves comfortable on the WattBike™prior to the commencement of their 10-min self-selected warm-up. Participants wore exercise clothing in which they felt most comfortable; this was shorts or exercise tights, lightweight t-shirt and shoes. The esti- mated insulative values of environment appropriate sportswear for males is0.45–0.61 clo and 0.57 clo for females [30,31,49]. Once settled on the bike prior to warm up, resting blood lactate was taken via a ngertip sample (Lactate Pro 2, Arkray Ltd., Koka-shi, Shiga, Japan). Heart rate (Polar RS400, Polar Electro Oy, Kempele, Finland), Trec, RPE, thermal comfort (TC) and thermal sensation (TS) were also recorded, following the mouth rinse and tting of the gas collection mask.

females [30,31,49]. Once settled on the bike prior to warm up, resting blood lactate was taken via a ngertip sample (Lactate Pro 2, Arkray Ltd., Koka-shi, Shiga, Japan). Heart rate (Polar RS400, Polar Electro Oy, Kempele, Finland), Trec, RPE, thermal comfort (TC) and thermal sensation (TS) were also recorded, following the mouth rinse and tting of the gas collection mask. VO2, VE and RER were then recorded continuously for the remaining duration of the protocol. The mouth rinse was performed approximately one minute prior to the completion of a modi ed three-minute all-out maximal test, for which the protocol was obtained via the Watt Bike™online manual (“The 3 Minute Aerobic Test”, 2021). Participants were asked to cycle `all out' for three minutes. No visual or verbal feedback was provided throughout the test and the participants were blinded to the Wattbike™screen, so data did not act as an external cue. At the 90 s mark and immediately following the test participants were asked to state their measure of TC, TS and RPE. Physiological respiratory measures were recorded continuously throughout the exercise period and into participant recovery, unless otherwise stated. Figure 2.3.1. Physiological Measures During the trials, a Metalyzer 3B CPET device (Coretex Medical, Leipzig, Germany), was used to analyse expired air throughout the duration of the warmup and the three- minute maximal test. Measures recorded were absolute and relative oxygen uptake (VO2; L/min and mL/kg/min), minute ventilation (VE; L/min) and respiratory exchange ratio (RER). Heart rate was recorded via a chest strap (Polar H10, Polar Electro Oy, Kempele Finland) using the Polar team app on a paired iPad (Apple, Los Altos, CA, USA). The rectal thermistor was connected to a data logger (SQ2010, Grant Instruments, Cambridge, UK) with values being recorded pre and post warm up as well as post mouth rinse, halfway through the maximal test and twice during the 5 min recovery period. Blood lactate was taken using a lancet to pin prick the participant's chosen nger to extract a capillary sample. The blood was collected using a lactate strip attached to a Lactate Pro 2 (Arkray Lactate Pro

The use of menthol (0.1%) as a mouth rinse showed small performance benefits for short duration high intensity exercise in the heat.