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This study examined the effect of honey supplementation on exercise performance and biochemical markers in comparison to plain water and sports drink in the heat. Ten recreational athletes (Age: 22.2 ± 2.0 years, weight: 65.7 ± 5.3 kg; height: 170.4 ± 3.5 cm; VO 2max : 51.5 ± 3.7 mL.kg −1 .min −1 ) participated in this study. Participants ran at 70% of their VO 2max for 1 h in a pre-load phase (Run-1), followed by a rehydration phase for 2 h and then a 20 min self-paced time trial (Run-2). After Run-1, participants drank either Acacia honey, sports drink or plain water with an amount equivalent to 150% of body weight loss. Subsequently, the participants performed the 20 min self-paced time trial. Acacia honey elicited an improved running time trial performance with a significantly (p<0.05) longer distance ran compared to plain water trial, but it was not different from the sports drink trial. In addition, there was no significant difference in running performance between sports drink and plain water trials. Plasma glucose, insulin and free fatty acids were significantly (p<0.05) higher in H and sports drink compared to the W trial during the rehydration phase. There were no significant differences in body weight changes, oxygen uptake, heart rate, rate of perceived exertion, tympanic temperature, plasma volume changes, plasma cortisol, urine osmolality, volume, and specific gravity among the three trials. Thus, Acacia honey can be recommended to be used as a rehydration drink for individuals who train and compete in the heat. Keywords: Acacia honey; heat; rehydration; running performance; urine osmolality ABSTRAK Penyelidikan ini mengkaji kesan madu sebagai diet tambahan terhadap prestasi senaman dan penanda biokimia berbanding dengan air kosong dan minuman sukan dalam persekitaran panas. Sepuluh atlet rekreasi (Umur: 22.2 ± 2.0 tahun; berat: 65.7 ± 5.3 kg;

as a rehydration drink for individuals who train and compete in the heat. Keywords: Acacia honey; heat; rehydration; running performance; urine osmolality ABSTRAK Penyelidikan ini mengkaji kesan madu sebagai diet tambahan terhadap prestasi senaman dan penanda biokimia berbanding dengan air kosong dan minuman sukan dalam persekitaran panas. Sepuluh atlet rekreasi (Umur: 22.2 ± 2.0 tahun; berat: 65.7 ± 5.3 kg; ketinggian: 170.4 ± 3.5 sm; VO 2max : 51.5 ± 3.7 mL.kg −1 .min −1 ) mengambil bahagian dalam kajian ini. Para peserta berlari pada 70% VO 2max mereka selama 1 jam dalam fasa pra-muat (Larian-1), diikuti dengan fasa rehidrasi selama 2 jam dan kemudian larian rentak-sendiri selama 20 min (Larian-2). Selepas Larian-1, peserta meminum madu Acacia, minuman sukan atau air kosong dengan jumlah yang setara dengan 150% penurunan berat badan. Selepas itu, para peserta melakukan percubaan masa rentak-sendiri selama 20 min. Madu Acacia menghasilkan prestasi larian percubaan masa yang lebih baik dan jarak yang lebih jauh secara signifikan (p<0.05) berbanding dengan percubaan air kosong. Namun, tidak ada perbezaan yang signifikan dalam prestasi larian antara percubaan minuman sukan dan air kosong. Glukosa, insulin dan asid lemak bebas plasma dalam percubaan madu Acacia dan minuman sukan adalah lebih tinggi secara signifikan (p<0.05) berbanding dengan percubaan air kosong ketika fasa rehidrasi. Tidak ada perbezaan yang signifikan dalam perubahan berat badan, pengambilan oksigen, denyutan jantung, kadar persepsi keletihan, suhu timpanik, perubahan isi padu plasma, kortisol plasma, keosmololan, isi padu dan nilai graviti khusus air kencing antara ketiga-tiga percubaan. Madu Acacia menghasilkan prestasi percubaan masa yang lebih baik berbanding dengan percubaan air tetapi tidak berbeza dengan minuman sukan. Justeru, madu Acacia mungkin berpotensi digunakan sebagai minuman rehidrasi bagi individu yang berlatih dan bertanding dalam persekitaran panas. Kata kunci: Haba; madu Acacia; osmolaliti air kencing; prestasi larian; rehidrasi

2294 INTRODUCTION Athletes perform physical activities such as training and competing through a range of environmental conditions which include hot and humid environments. Athletes who train in the heat on a daily basis are predisposed to a high risk of dehydration. Hence, proper rehydration is recommended for these individuals to avoid any adverse effects of dehydration. The main aim for adequate rehydration is for restoration of muscle and liver glycogen as well as replacement of body water and electrolytes (Lopez 2012). During the recovery phase, ingestion of carbohydrate is important to replenish the muscle and liver glycogen. Glycogen is a readily available source of energy for the muscles, and it is crucial for refueling performance during training or competition. The purpose of consuming high carbohydrate drink is to ensure adequate carbohydrate intake and to replenish carbohydrate stores after exhaustive exercise (Jeukendrup 2004). Glycogen depletion and hypoglycemia have been associated with fatigue and decrement in the performance during prolonged exercise (Tsintzas & Williams 1998). Ingestion of fluid containing carbohydrate is recommended to improve exercise performance as muscle glycogen storage is replenished, and the decline in blood glucose and dehydration can be avoided (Ivy 1999; Murray 1996). Consuming fluid in direct proportion to sweat loss during rehydration could maintain proper physiological functions and has been shown to significantly improve exercise performance (James et al. 2017). Additionally, restoration of body fluid losses is necessary for optimal cardiovascular function and thermoregulation during subsequent exercise (Takamata et al. 1998). The risks of dehydration on adverse consequences on sports performance such as impaired endurance exercise performance indicated by a shortened time to exhaustion or a reduction in sustainable exercise intensity are well- documented (Casa et al. 2019). Thus, various types of drinks have been used as rehydration strategies during post-exercise to ensure adequate recovery before the next bout of exercise. These drinks include deep-ocean mineral water (Keen et al. 2016), maple water (Matias et al. 2019) and yogurt drink (Niksefat et al. 2019). Sports drink usually contains 4 to 8% carbohydrate and electrolytes (Murray 1996). Fluid and electrolyte replacement promote proper rehydration which is important in

rehydration strategies during post-exercise to ensure adequate recovery before the next bout of exercise. These drinks include deep-ocean mineral water (Keen et al. 2016), maple water (Matias et al. 2019) and yogurt drink (Niksefat et al. 2019). Sports drink usually contains 4 to 8% carbohydrate and electrolytes (Murray 1996). Fluid and electrolyte replacement promote proper rehydration which is important in delaying the onset of fatigue either during exercise, training, or competition (Adams et al. 2019; Casa et al. 2019). The composition of honey depends primarily on the floral sources. It is a supersaturated sugar which is mainly composed of a complex mixture of carbohydrate and gets its sweetness from the monosaccharides i.e., fructose and glucose, and has approximately the same relative sweetness as that of granulated sugar (Saxena et al. 2010). With respect to carbohydrates, honey contains mainly fructose (about 38.5%) and glucose (about 31.0%). Honey also contains trace amounts of several vitamins and minerals. It also contains tiny amounts of several compounds such as vitamin C which functions as antioxidants (Gheldof et al. 2002; Martos et al. 2000). Earnest et al. (2004) investigated the effect of honey gel supplementation during a 64 km cycling time trial performance. In this study, nine cyclists ingested a gel containing honey, dextrose, and flavored placebo with 250 mL of water at every 16 km during a 64 km time trial. Their results showed that honey and dextrose ingestion elicited shorter cycling time to complete 64 km compared to placebo. They concluded that honey could serve as a useful alternative form of carbohydrate for enhancing sport performance in athletes. Another study had also compared honey drink with plain water, and it was found that rehydration with honey drink has potential to improve endurance running performance (Ahmad et al. 2015). Nevertheless, to date, study on the comparison between honey drink and a sports drink as a rehydration aid has not been carried out. The purpose of this study was to investigate the effects of honey supplementation on time trial performance and selected physiological parameters compared to a commercially-available sports drink and plain water in the

performance (Ahmad et al. 2015). Nevertheless, to date, study on the comparison between honey drink and a sports drink as a rehydration aid has not been carried out. The purpose of this study was to investigate the effects of honey supplementation on time trial performance and selected physiological parameters compared to a commercially-available sports drink and plain water in the heat. We hypothesized that there was a significant difference in subsequent running performance with post-exercise ingestion of honey drink compared to a commercially-available sports drink or plain water. MATERIALS AND METHODS PARTICIPANTS Ten recreational male athletes (20 to 30 years old) who performed regular exercise for at least 30 min per session and more than two times per week (minimum duration of 2 months prior to the study) were recruited. Sample size was calculated by Power and Sample size calculation software (Version 3.0) based on Ooi et al. (2001). Power of the study was set at 80% with 95% confidence interval.

2295 Standard deviation observed was 1.67 and difference in population mean was 1.5, therefore the effect size (d) used was 0.89. The calculated sample size was 10 participants per group. All the participants were able to run on motorized treadmill at 70% of their respective maximal oxygen uptake (VO 2max ) for at least 60 min. Participants who fulfilled the inclusion criteria provided their written informed consent. The exclusion criteria were individuals having any acute and chronic diseases such as asthma, stroke, diabetes and heart problems. This counter-balanced, crossover study was approved by the Human Research Ethics Committee, Universiti Sains Malaysia (USMKK/ PPP/JEPeM [239.3(14)]). PRELIMINARY TRIALS This study was conducted at the Exercise and Sports Science Laboratory, School of Health Sciences, Universiti Sains Malaysia. The following preliminary tests were conducted before the main experimental trials: A 16 min incremental sub-maximal running test to establish the relationship between running speed and oxygen uptake (VO 2 ); and A graded maximal treadmill running test to exhaustion to determine each participant’s VO 2max (Keong et al. 2006; Muhammad et al. 2018; Wee et al. 1999). SUB-MAXIMAL TEST Prior to this test, participants were fitted with heart rate monitor, head gear, mouth piece and nose clip. The participants were required to run on the treadmill with an initial speed set at 6 km.h −1 . At the end of each 4 min stages, the speed was increased by 1 km.h −1 until the speed of 9 km.h −1 was achieved. Expired air was analyzed for oxygen consumption via gas analyzer (VMax Spectra, USA). In addition, heart rates were also recorded throughout the test. The oxygen uptake during the final minute of each 4 min increment was recorded. Then, the running speed for the warm-up at 50% VO 2max and endurance running performance at 70% VO 2max for each participant was estimated from the plot of a graph with oxygen consumption versus the four speeds ran during this submaximal test (Wilmore et al. 1998). MAXIMAL OXYGEN UPTAKE (VO 2max ) TEST The VO 2max test required the participants to run until exhaustion at a constant

at 50% VO 2max and endurance running performance at 70% VO 2max for each participant was estimated from the plot of a graph with oxygen consumption versus the four speeds ran during this submaximal test (Wilmore et al. 1998). MAXIMAL OXYGEN UPTAKE (VO 2max ) TEST The VO 2max test required the participants to run until exhaustion at a constant speed while the inclination was increased. During the test, inclination of treadmill was increased by 2.5° for every 3 min starting from the initial inclination of 3.5°. The set up for the participants was similar with the sub maximal test. At every 20 s, the expired air was analyzed by the gas analyzer (VMax Spectra, USA). Heart rate and rate of perceive exertion (RPE) were also taken during the last 20 s of each incremental stage. The test was terminated when participants could no longer run despite verbal encouragement. The value of VO 2max was accepted when the participants achieved 3 out of the following 4 criteria: a VO 2 leveling off; Respiratory exchange ratio of more than 1.15; heart rate more that 95% of age-predicted HR max ; and RPE of more than 18 Borg’s unit (Edvardsen et al. 2014). FAMILIARIZATION WITH THE EXPERIMENTAL TRIALS A familiarization trial was carried out to familiarize the participants with the intensity and environmental condition of the main experimental trials. Participants were required to run on the treadmill at 70% of their respective VO 2max for 60 min in the heat (31 °C, 70% relative humidity). Running speed during warm up and endurance running was determined from the data obtained from the preliminary trials. Participants who were unable to run at 70% of their VO 2max for at least 60 min were excluded from the study. HONEY AND SPORTS DRINK Acacia honey which was used in the present study originated from the state of Johor in Malaysia. Acacia honey is produced by bees known as Apis mellifera which collects their nectar from wild Acacia leaves (Khalil et al. 2011). Based on the analysis made by the Laboratory of Department of Molecular Medicine, Universiti

from the study. HONEY AND SPORTS DRINK Acacia honey which was used in the present study originated from the state of Johor in Malaysia. Acacia honey is produced by bees known as Apis mellifera which collects their nectar from wild Acacia leaves (Khalil et al. 2011). Based on the analysis made by the Laboratory of Department of Molecular Medicine, Universiti Malaya, Acacia honey used in the present study contains fructose, glucose, sucrose, and maltose. Each 100 mL of Acacia honey contains 302 kcal of energy, 75 g carbohydrate, 31.2 g fructose, 22.9 g glucose, 9.9 g sucrose, 3.3 g maltose, 13 mg sodium and 0.5 g protein (Ahmad et al. 2020). The commercially available sports drink contains 20 kcal energy, 4.9 g carbohydrate, 42 mg sodium, 19 mg potassium, 4 mg calcium, 40 mg chloride and 17 mg phosphate for every 100 mL. MAIN EXPERIMENTAL TRIALS The participants were required to perform three main trials (Run-2) after ingestion of either honey drink,

2296 sports drink or plain water during the rehydration period. These three trials were counter-balanced and carried out one week apart. On each day of the experimental trials, participants reported to the laboratory after 10 h of overnight fast. The following procedures were carried out just before the commencement of the trial: A standardized breakfast with one piece of bread and 500 mL of plain water; Measurement of nude body mass; Urine sample collection; Cannulation at the forearm vein to make repeated withdrawal of blood samples. To minimize differences in resting muscle glycogen concentrations between trials, participants recorded their food intake for three days prior to the first experimental trial in a food diary. They were then instructed to follow the same diet before the second and third trials. PRE-LOAD PHASE (RUN-1) For Run-1, the participants were required to run on a treadmill in the heat (31 °C, 70% relative humidity) at 70% of their respective VO 2max for 60 min. Before the commencement of the run, participants were fitted with heart rate monitor. Blood sample was collected, resting heart rate, tympanic temperature and expired air were measured prior to the warm-up of Run-1. Then, the participants warmed up for 5 min by running at 50% of their respective VO 2max . Blood sample was collected at the end of warm up. After the warmup, the intensity was increased to 70% of their respective VO 2max . At intervals of 20 min during Run-1, expired air samples, heart rate, tympanic temperature, room temperature and relative humidity were recorded. At the end of the 60 min run, blood sample was collected and expired air, heart rate and tympanic temperature were recorded. Then, post-exercise nude body weight was obtained after the participant had towel-dried themselves. REHYDRATION PHASE After completion of Run-1, the participants were required to rest for 2 h. During this rehydration phase, plain water or honey drink or sports drink was consumed by the participants in a randomized fashion (counter- balanced). The amount of fluid which was ingested by the participants was equivalent to 150% of body weight loss (Lee

had towel-dried themselves. REHYDRATION PHASE After completion of Run-1, the participants were required to rest for 2 h. During this rehydration phase, plain water or honey drink or sports drink was consumed by the participants in a randomized fashion (counter- balanced). The amount of fluid which was ingested by the participants was equivalent to 150% of body weight loss (Lee et al. 2011; Maughan et al. 1996) in 3 boluses of 60%, 50%, and 40% consecutively (Ahmad et al. 2020; Saat et al. 2002). Ingestion of the drink was at 0, 30, and 60 min during the rehydration phase. Blood and urine samples were also taken at every 30 min during this period. 20 MIN SELF-PACED TIME TRIAL PERFORMANCE (RUN-2) The participants were then required to perform a 20 min self-paced running time trial (Run-2), where the longest distance covered in 20 min was used to indicate endurance running performance. Other studies have also used self-paced time trial to determine exercise performance (Bradbury et al. 2019; Holgado et al. 2019; McGawley 2017; Ping et al. 2018). The speed of running during the time trial was self-paced by the participants via a speed controller. Blood sample, expired air, heart rate, tympanic temperature, and RPE were taken at the end of the time trial. Urine sample was also collected after completing the time trial. Then, post- exercise nude body weight was obtained after participants has towel- dried themselves. BLOOD ANALYSIS Eight mL of blood was collected during each blood withdrawal. One mL of the blood was transferred into an EDTA (Ethylenediamine tetra-acetic acid) tube. It was used to measure the hematocrit levels using the capillary method via a micro hematocrit reader (Hawksley, England). The plasma volume changes were calculated from the results of these hematocrit levels using the formula of van Beaumont et al. (1981). The rest of the blood was transferred into another tube containing natrium fluoride as the anticoagulant. After centrifugation at 3000 rpm for 10 min at 4 °C, the plasma was transferred into a 1.5 mL tube and stored at -40 °C for subsequent analysis of glucose, cortisol,

of these hematocrit levels using the formula of van Beaumont et al. (1981). The rest of the blood was transferred into another tube containing natrium fluoride as the anticoagulant. After centrifugation at 3000 rpm for 10 min at 4 °C, the plasma was transferred into a 1.5 mL tube and stored at -40 °C for subsequent analysis of glucose, cortisol, insulin, and free fatty acid. Plasma glucose concentration was determined by the enzymatic oxidation method (Randox, United Kingdom) while insulin concentrations were determined using a spectrophotometer (Spekol 200, Germany). Plasma cortisol levels were measured using an ELISA kit (Human, Germany) while plasma free fatty acid concentrations were determined by acylcoenzyme- A-synthetase (ACS) and acyl-coenzyme-A-oxidase (ACOD) method (Wako, Japan). Intra-assay coefficients of variation (CV) for glucose, cortisol, insulin and free fatty acid were 5.7, 5.2, 2.9, and 7.9%, respectively. URINE ANALYSIS Urine volume was measured using a measuring cylinder, and urine osmolality was measured by using cryscopic osmometer (Osmomat 030, Gonotec, Germany). Urine specific gravity was measured using a refractometer

2297 (Atago, SPR-T2, Japan). These parameters were measured to indicate the participants’ rehydration status during the rehydration phase. STATISTICAL ANALYSIS Changes in physiological responses, hematological, and urine parameters were analyzed using a two-way ANOVA with repeated measures. When appropriate, Bonferroni post hoc analyses were used to examine differences between trials. The 20 min self-paced time trial performance with all three drinks were analyzed using one-way ANOVA. The Statistical Package for Social Sciences (SPSS) program version 22.0 was used for statistical analysis. Differences were considered significant at p<0.05. Results are presented as means ± SD. RESULTS PARTICIPANT’S PHYSIOLOGICAL CHARACTERISTICS Ten healthy male recreational runners completed this study. Their age, body weight, height, body mass index, and VO 2max are 22.2 ± 1.7 years, 65.7 ± 5.3 kg., 170.4 ± 3.5 cm, 22.7 ± 1.6 kg.m -2 and 51.5 ± 3.7 mL.kg −1 .min −1 , respectively. In all the three trials, room temperature and relative humidity were well-maintained at 31.4 °C and 70.4%, respectively (Table 1). TABLE 1. Room temperature, relative humidity, body weight changes in plain water (W), honey (H) and sports drink (SPD) trials during Run-1 and Run-2 Variables W trial H trial SPD trial Room temperature (°C) 31.4 ± 0.2 31.4 ± 0.2 31.4 ± 0.1 Relative humidity (%) 70.4 ± 0.1 70.4 ± 0.2 70.4 ± 0.2 Pre Run-1 body weight (kg) 65.7 ± 5.0 65.5 ± 4.7 65.5 ± 5.1 Post Run-1 body weight (kg) 64.4 ± 4.8 64.1 ± 4.6 64.1 ± 5.0 Body weight changes after Run-1 (%) 2.0 ± 0.4 2.1 ± 0.3 2.1 ± 0.3 Pre Run-2 body weight (kg) 65.4 ± 5.0 65.3 ± 4.7 65.3 ± 5.3 Post Run-2 body weight (kg) 64.8 ± 4.3 64.7 ± 4.6 64.6 ± 5.2 Body weight changes after Run-2 (%) 1.0 ± 0.3 1.0 ± 0.2 1.0 ± 0.4 Body weight of the participants at Pre-Run-1 and Post Run-1 were not significantly different among the three trials (Table 1). Percent body weight loss after Run 1 was 2.0% or more and it was not different among the three trials. Similarly, body weight of