Effect of Glycerol-Induced Hyperhydration on a 5-kilometer Running Time-Trial Performance in the Heat in Recreationally Active Individuals

mal oxygen consumption ( . V O 2max) is a major determinant of 5-km running time-trial (TT) performance. Glycerol-induced hyperhydration (GIH) could improve . V O 2maxin recreationally active persons through an optimal increase in plasma volume. Moreover, ingestion of a large bolus of cold uid before exercise could decrease thermal stress during exercise, potentially contributing to improved performance. We determined the effect of GIH on 5-km running TT performance in 10 recreationally active individuals (age: 24 4 years; . V O 2max: 48 3 mL/kg/min). Using a randomized and counterbalanced protocol, participants underwent two, 120-min hydration protocols where they ingested a (1) 30 mL/kg fat-free mass (FFM) of cold water (~4 C) with an arti cial sweetener + 1.4 g glycerol/kg FFM over the rst 60 min (GIH) or (2) 7.5 mL/kg FFM of cold water with an arti cial sweetener over the rst 20 min (EUH). Following GIH and EUH, participants underwent a 5-km running TT at 30 C and 50% relative humidity. After 120 min, GIH was

water (~4 C) with an arti cial sweetener + 1.4 g glycerol/kg FFM over the rst 60 min (GIH) or (2) 7.5 mL/kg FFM of cold water with an arti cial sweetener over the rst 20 min (EUH). Following GIH and EUH, participants underwent a 5-km running TT at 30 C and 50% relative humidity. After 120 min, GIH was associated with signi cantly greater uid retention (846 415 mL) and plasma volume changes (10.1 8.4%) than EUH, but gastrointestinal (GI) temperature did not differ. During exercise, 5-km running TT performance (GIH: 22.95 2.62; EUH: 22.52 2.74 min), as well as heart rate, GI temperature and perceived exertion did not signi cantly differ between conditions. This study demonstrates that the additional body water and plasma volume gains provided by GIH do not improve 5-km running TT performance in the heat in recreationally active individuals. Keywords:hyperhydration; hydration; glycerol; performance; running 1. Introduction Maximal oxygen consumption ( . V O2max) is a determinant factor of 5-km running time- trial (TT) performance. Indeed, Ramsbottom et al. [1] demonstrated that a 5-km running TT performance can be explained, at least in large part, by the magnitude of . V O2max. This observation is unsurprising, as an all-out 5-km running TT is completed at approximately 90% of . V O2maxand 98% of maximum heart rate [2]. The Fick equation dictates that the . V O2maxdepends on both the cardiac output and the arteriovenous oxygen difference [3]. Therefore, it is reasonable to believe that any positive alteration in . V O2max, either by manipulating cardiac output, or arteriovenous oxygen difference, or both, could potentially lead to an improved 5-km running TT performance. It is possible to increase cardiac output by arti cially increasing plasma volume [4]. A simple and legal way to enhance plasma volume is to induce hyperhydration prior to exercise using glycerol. Indeed, glycerol- induced hyperhydration (GIH) has been shown to increase total body water by ~800 mL and plasma volume by ~8% [5–7] 2 h following the ingestion of 26 mL of water/kg body mass (BM) with 1.2 g of glycerol/kg BM. Nutrients2023,15,

A simple and legal way to enhance plasma volume is to induce hyperhydration prior to exercise using glycerol. Indeed, glycerol- induced hyperhydration (GIH) has been shown to increase total body water by ~800 mL and plasma volume by ~8% [5–7] 2 h following the ingestion of 26 mL of water/kg body mass (BM) with 1.2 g of glycerol/kg BM. Nutrients2023,15, 599.

Nutrients2023,15, 599 2 of 15 The capacity of an increase in plasma volume to enhance . V O2maxappears to be dependent upon an individual's level of training and the magnitude of the increase in plasma volume [8]. Indeed, in general, this blood manipulation has been shown to be advantageous (1) in sedentary or recreationally trained individuals [9] and (2) when an increase in plasma volume of the order of 7–8% or 200–300 mL is generated [10]. The plasma volume of healthy people weighing 60–75 kg with an average hematocrit of 40% is estimated to be approximately 2520–3150 mL [11]. Therefore, an increase in plasma volume of 8% in this population would generate a theoretical increase in plasma volume of the order of 200 to 250 mL. Thus, it is reasonable to believe that the use of pre-exercise GIH could lead to an increase in . V O2max. In this regard, Patlar et al. [12] demonstrated that GIH improves . V O2maxboth in sedentary and exercising individuals, likely through an optimal augmentation of cardiac output as there is no reason to believe that, mechanistically, GIH could widen arteriovenous oxygen difference. In addition, decreased hyperthermia during exercise may help endurance perfor- mance [13]. To this effect, at any given exercise intensity during exercise conducted under warm ambient conditions, an increased plasma volume may lead to a more favorable distribution of blood to the working muscles and skin, thereby favoring heat dissipation and improved performance [14]. On the other hand, the ingestion of 26 mL of water/kg BM provided at 4 C would be expected to result in a drop in core body temperature, compared to a pre-exercise euhydration state [15,16]. All observations provided above lead us to believe that GIH may be advantageous for endurance performance under warm ambient conditions. Therefore, the purpose of this study was to compare the effect of pre-exercise GIH to a state of pre-exercise euhydration (EUH) on uid balance responses, performance, gastrointestinal (GI) temperature, heart rate and rating of perceived exertion during a 5-km running TT conducted in a warm environment in recreationally active individuals. We hypothesized

advantageous for endurance performance under warm ambient conditions. Therefore, the purpose of this study was to compare the effect of pre-exercise GIH to a state of pre-exercise euhydration (EUH) on uid balance responses, performance, gastrointestinal (GI) temperature, heart rate and rating of perceived exertion during a 5-km running TT conducted in a warm environment in recreationally active individuals. We hypothesized that (1) GIH would produce a state of hyperhydration of at least 800 mL; (2) the increase in plasma volume at the beginning of exercise would be more important and its decline less important immediately following exercise with GIH than EUH; (3) there would be no signi cant difference in heart rate and rating of perceived exertion during the 5-km running TT between conditions; (4) the GI temperature would be lower with GIH than EUH throughout the hydration and exercise periods; and (5) the 5-km running TT time would be faster with GIH than EUH. 2. Materials and Methods 2.1. Participants Ten (9 men; 1 woman) recreationally active individuals (mean SD: 24 4 years; 175 10 cm; 69.5 9.3 kg; 13.6 6.7% body fat; 60.3 9.4 kg fat-free-mass (FFM); 190 9 b pm for maximal heart rate; 48 3 mL/kg/min for . V O2max) participated in this study. Inclusion criteria were: (1) being healthy and between the age of 18–50 years, (2) training at least 3 h per week, (3) practicing an aerobic sport for at least one year, (4) takingno medication that can affect core body temperature and hydration state and (5) having a body mass index < 30 kg/m 2 . The protocol was thoroughly explained, and participants gave their written informed consent to participate in this study. The CIUSSS Estrie-CHUS Ethics Committee (2020-3606) approved all experimental procedures. 2.2. Overview of the Study The study used a randomized and counterbalanced protocol. Participants rst under- went a preliminary visit where baseline measurements were taken. No more than 10 days following the rst visit, participants realized a familiarization trial to customize themselves with the 5-km running TT. Participants then underwent two experimental trials (GIH and EUH) separated by 7 days

2.2. Overview of the Study The study used a randomized and counterbalanced protocol. Participants rst under- went a preliminary visit where baseline measurements were taken. No more than 10 days following the rst visit, participants realized a familiarization trial to customize themselves with the 5-km running TT. Participants then underwent two experimental trials (GIH and EUH) separated by 7 days and conducted at the same time of day.

Nutrients2023,15, 599 3 of 15 2.3. Preliminary Visit During the preliminary visit, the height of the participants, wearing only socks, was measured to the nearest 0.5 cm using a wall stadiometer, post-void nude BM with a digital scale (BX-300+, Altron Systems, Mount Pleasant, SC, USA, 20 g), fat mass and FFM with the dual-energy X-ray absorptiometry technology (Lunar Prodigy, GE Healthcare, Chicago, IL, USA) and blood pressure and resting heart rate using a digital sphygmomanometer (Welch-Allyn 420 series, Skaneateles Falls, NY, USA) after the participants remained seated for a period of 2 min. Finally, . V O2maxwas determined with a metabolic analyzer (Cosmed Quark CPET, Cosmed, Chicago, IL, USA) calibrated according to the manufacturer instruc- tions using an incremental running protocol on a motorized treadmill. Participants started walking at 5 km/h with 0% grade for 1 min, with further increments of 1 km/h occurring every 1 min until the participants could not continue. The . V O2maxwas con rmed when at least two of those criteria were reached: (1) respiratory exchange ratio 1.1, (2) theo- retical maximal heart rate (220-age), (3) . V O2plateau concurrent to an increase in running speed [17]. 2.4. Pre-Experimental Protocol For the 24-h period preceding the familiarization trial, participants lled a dietary log (nutrition and hydration), which they replicated for the 24-h period preceding the two experimentations. Participants refrained from consuming diuretics, with the exception of caffeine, during the 24-h period preceding the experimentations as well as from taking any supplements in the 48-h period preceding the experimentations [5–7]. To optimize hydration before the experimentations and familiarization trial, participants drank 250 mL of water 120 min before going to sleep the night before these visits and 250 mL 60 min before arriving at the laboratory [5–7]. Both before the experiments and the familiarization trial, participants refrained from eating and drinking in the 60-min period preceding their arrival at the laboratory, went to bed at the same time of day the preceding nights and maintained their training routine over the last 24-h period, while stopping any form of exercise for the last 8 h leading to

[5–7]. Both before the experiments and the familiarization trial, participants refrained from eating and drinking in the 60-min period preceding their arrival at the laboratory, went to bed at the same time of day the preceding nights and maintained their training routine over the last 24-h period, while stopping any form of exercise for the last 8 h leading to these visits [7]. 2.5. Familiarization Trial A familiarization trial was executed to minimize any learning effect [18]. During this visit, participants underwent the 5-km running TT under the same experimental conditions as those used during the experiments, with the exception that they were not required to ingest the GI pill or to undergo the hydration protocols beforehand. 2.6. Experimental Trials Each experimental trial was divided into three phases: (1) arrival at the laboratory and baseline data collection, (2) 120-min hydration period and, (3) 5-km running TT. Figure participants voided their bladder in a graded urinal. Participants were then weighed and instrumented with a heart rate monitor after which they were seated for 10 min with their hand immersed in 40 C water. A capillary blood punction was then performed and measurements of heart rate, GI temperature and subjective perceptions were taken. Then, the participants started the 120-min hydration period, which consisted of in- gesting either (1) 7.5 mL/kg FFM of cold water (~4 C) with an arti cial sweetener + 1.4 g glycerol/kg FFM at times 0, 20, 40 and 60 (GIH) [5–7] or (2) 7.5 mL/kg FFM of cold water with an arti cial sweetener at time 0 only (EUH). Every 20 min up until min 120, heart rate, GI temperature and subjective perceptions were measured and then participants urinated, urine was collected and BM was measured. With the exception of when they urinated and were weighed, participants always remained seated. Capillary blood samples were collected at minutes 60 and 120; they occurred prior to standing to limit the impact of body posture on plasma volume changes and following 10 min of hand immersion in 40 C

exception of when they urinated and were weighed, participants always remained seated. Capillary blood samples were collected at minutes 60 and 120; they occurred prior to standing to limit the impact of body posture on plasma volume changes and following 10 min of hand immersion in 40 C

Nutrients2023,15, 599 4 of 15 water. Skin thermistors were installed on participants over the last 20-min period of the hydration period.Nutrients 2023, 15, x FOR PEER REVIEW 4 of 15 urinated, urine was collected and BM was measured. With the exception of when they urinated and were weighed, participants always remained seated. Capillary blood sam- ples were collected at minutes 60 and 120; they occurred prior to standing to limit the impact of body posture on plasma volume changes and following 10 min of hand immer- sion in 40 °C water. Skin thermistors were installed on participants over the last 20-minute period of the hydration period. Figure 1. Illustration of the experimental protocol. Following the hydration period, participants were transferred to the environmental chamber where they mount the motorized treadmill and remained silent for 2 min. Then, the participants ran at a pace of their choice on the treadmill for a 5-minute period to warm up before the TT. The participants’ heart rates, GI and skin temperatures and subjective perceptions were measured before and after the warmup and then the 5-kilometer run- ning TT started at 0% grade, after the participants had recovered for 2 min. The partici- pants could adjust their speed throughout the running period and did not have access to any metrics, such as speed and elapsed time, except for the distance completed. Standard- ized verbal encouragement was provided during the TT. Two propeller-type fans (24″ diameter, 8800 CFM, Secco international, St-Hyacinthe, QC, Canada) as well as an axial- type fan (13″ diameter, 1720 CFM, Maximum Canada, Toronto, ON, Canada) simulated wind in front of participants at a velocity closely approximating running speed. Wind speed was verified using an anemometer (DAF800, General Tools & Instruments, Secau- cus, NJ, USA). Heart rate, GI, skin temperatures and subjective perceptions were meas- ured at the end of each km. A capillary blood sample was taken immediately following the TT. Fluid was not provided during exercise. 2.7. Measurements 2.7.1. Heart Rate and Gastrointestinal and Skin Temperatures Heart rate was measured with a Garmin Premium heart rate monitor (Garmin, Olathe, KS, USA).

NJ, USA). Heart rate, GI, skin temperatures and subjective perceptions were meas- ured at the end of each km. A capillary blood sample was taken immediately following the TT. Fluid was not provided during exercise. 2.7. Measurements 2.7.1. Heart Rate and Gastrointestinal and Skin Temperatures Heart rate was measured with a Garmin Premium heart rate monitor (Garmin, Olathe, KS, USA). Gastrointestinal temperature was measured using calibrated telemetric pills (CoreTemp, Palmetto, FL, USA) ingested 10 h prior to the participants’ arrival at the laboratory [19]. Skin temperature was measured with calibrated YSI 409 B probes (Yellow Springs Instrument, Yellow Springs, OH, USA) placed on the left side of the body on the chest, the forearm, the thigh and the calf. Hypafix dressing tape was used to hold the probes in place. The skin probes were connected to a USB-TEMP data acquisition box (MC measurement computing, Norton, MA, USA). Mean skin temperature was calculated ac- cording to Ramanathan [20] using the following Equation (1): Tskin = (0.3 × Tchest) + (0.3 × Tforearm) + (0.2 × Tthigh) + (0.2 × Tcalf), (1) Figure 1.Illustration of the experimental protocol. Following the hydration period, participants were transferred to the environmental chamber where they mount the motorized treadmill and remained silent for 2 min. Then, the participants ran at a pace of their choice on the treadmill for a 5-min period to warm up before the TT. The participants' heart rates, GI and skin temperatures and subjective perceptions were measured before and after the warmup and then the 5-km running TT started at 0% grade, after the participants had recovered for 2 min. The participants could adjust their speed throughout the running period and did not have access to any metrics, such as speed and elapsed time, except for the distance completed. Standardized verbal encouragement was provided during the TT. Two propeller-type fans (24 00 diameter, 8800 CFM, Secco international, St-Hyacinthe, QC, Canada) as well as an axial-type fan (13 00 diameter, 1720 CFM, Maximum Canada, Toronto, ON, Canada) simulated wind in front of participants at a velocity closely approximating running speed. Wind speed was

elapsed time, except for the distance completed. Standardized verbal encouragement was provided during the TT. Two propeller-type fans (24 00 diameter, 8800 CFM, Secco international, St-Hyacinthe, QC, Canada) as well as an axial-type fan (13 00 diameter, 1720 CFM, Maximum Canada, Toronto, ON, Canada) simulated wind in front of participants at a velocity closely approximating running speed. Wind speed was veri ed using an anemometer (DAF800, General Tools & Instruments, Secaucus, NJ, USA). Heart rate, GI, skin temperatures and subjective perceptions were measured at the end of each km. A capillary blood sample was taken immediately following the TT. Fluid was not provided during exercise. 2.7. Measurements 2.7.1. Heart Rate and Gastrointestinal and Skin Temperatures Heart rate was measured with a Garmin Premium heart rate monitor (Garmin, Olathe, KS, USA). Gastrointestinal temperature was measured using calibrated telemetric pills (CoreTemp, Palmetto, FL, USA) ingested 10 h prior to the participants' arrival at the laboratory [19]. Skin temperature was measured with calibrated YSI 409 B probes (Yellow Springs Instrument, Yellow Springs, OH, USA) placed on the left side of the body on the chest, the forearm, the thigh and the calf. Hypa x dressing tape was used to hold the probes in place. The skin probes were connected to a USB-TEMP data acquisition box (MC measurement computing, Norton, MA, USA). Mean skin temperature was calculated according to Ramanathan [20] using the following Equation (1): T skin= (0.3 T chest) + (0.3 T forearm) + (0.2 T thigh) + (0.2 T calf), (1) where T is temperature.

This study examines the effects of glycerol-induced hyperhydration on running performance.