This study was approved by the Institutional Human Research Ethics Committee, and complied with the Declaration of Helsinki. Ten young men were recruited for the present study, and each of them signed an informed consent form and completed medical questionnaires before participating in the study. The effect size for the difference in TF changes between conditions was estimated to be 0.9 based on our previous study , and 10 participants were shown to be adequate with the alpha level of 0.05 and power (1 − β) of 0.80. Their mean ± SD (range) age, height and body mass were 25.0 ± 2.7 (22–31) years, 173.7 ± 6.4 (165–184) cm and 74.0 ± 12.0 (57.2–89.3) kg, respectively.
All participants were in good health and fitness, participated in moderate exercise and sporting activities 2–3 times a week (less than 300 min in total), and were not prone to muscle cramping. However, participants responded to the electrical train stimulation described below and had muscle cramping in the screening. This was checked in a familiarization session that was set 7–10 days before the first experimentas session. If participants did not tolerate to the electrical stimulation, or no muscle cramping was induced by the electrical stimulation, they were excluded from the study. Thus, all participants included in the present study were muscle cramp responders to the electrical stimulation. In the familiarizarion session, the participants also experienced downhill running (DHR) with the slope of 5% for 10 min at 5.5–6.0 km·/h− 1. They were not exposed to temperature higher than 32 °C during the 4 weeks prior to this study.
The present study used OS-1 (Otsuka Pharmaceutical Factory, Inc., Japan) containing sodium (1150 mg/L = 50 mM/L), potassium (780 mg/L = 20 mM/L), magnesium (24 mg/L = 1 mM/L), chloride (1770 mg/L = 50 mM/L), glucose (18,000 mg/L = 100 mM/L) and others (e.g., phosphorus) as ORS. For the other condition, spring water (Coles Natural Spring Water, Coles, Australia) was used which contained a small amount of sodium (2 mg/L), potassium (0.5 mg/L), magnesium (18 mg/L), chloride (1.2 mg/L), and calcium (39 mg/L). The fluid ingested during and after DHR was the same for each condition. Using a cross-over design, the OS-1 and spring water conditions were compared for changes in TF of electrical stimulation to induce calf muscle cramp before and after DHR. The two conditions were counterbalanced among the participants and separated by a week. DHR was used in the present study, since its metabolic demand is smaller than that in level or uphill running . Thus, DHR was easier for the particiopants to perform, but induced relatively large sweating of nearly 2% of body mass in less than 60 min .
Muscle cramp assessment
To assess calf muscle cramp susceptibility, calf muscles were electrically stimulated to induce muscle cramping, and the frequency of the stimulation to induce muscle cramp was used as an indicator of muscle cramp susceptibility . Each participant lay prone on a massage bed, and the instep was placed on the bed, which kept the ankle joint in a plantar-flexed position. Electrical train stimulation was delivered to the calf muscles of the kicking (dominant) leg by a portable electrical stimulator (Compex 2, Compex Medical, Switzerland) with one electrode (cathode) placed over the tibialis posterior nerve in the popliteal fossa, and the other electrode (anode) placed at the tibialis tendon. The locations of the electrodes were marked by a semi-permanent marker to ensure the consistent electrode placement between measures on the same day and between sessions separated by a week. Each stimulation consisted of 0.5-s duration of rise time and 2-s bursts of stimuli of 300-μs duration, which was specifically programmed for the present study. The stimulation started at a frequency of 10-Hz, and two stimulations were given at this frequency during which the stimulation intensity was increased to a level (18–60 mA) which had been determined in a familiarisation session. The intensity of the stimulation was set for each participant to have muscle cramp at 24 or 26 Hz, and the same intensity was used in all measurements. This method was developed for the present study, based on our previous study . The intensity (amplitude) of the stimulation varied among participants (40–60 mA), but all of them had muscle cramping at 24 or 26 Hz at the baseline. The stimulation frequency was automatically increased by 2 Hz from 10 Hz every 30 s until muscle cramp was induced, and the TF at which cramp was induced was recorded. The muscle cramp was identified by a visibly taut muscle sustained after stimulation, and pain reported by the participant. Participants were instructed to relax during the electrical stimulation, and as soon as muscle cramp was confirmed, the cramp was relieved by passive dorsiflexion of the foot by the investigator.
All participants were instructed to refrain from any strenuous exercise for one week prior to participating in the study. They were asked to consume 600-ml of spring water at 2 h before coming to the laboratory, and refrain from any food and beverage intake thereafter. All participants were required to record their food intake before the first session, and they were asked to have the same foods and amount of water before the second session. However, the actual food and fluid intakes were not checked nor recorded, thus it was not not known whether the meal content and fluid intake before the two sessionas were identical.
The participants performed two bouts of DHR (slope: 5%) in a climate chamber at 35–36 °C and 25–28% relative humidity (Fig. 1). The running intensity and duration to reduce 2% of body mass (1.14–1.78 L) without fluid intake were based on the previous study . The running velocity was between 6.4–9.7 km/h among the participants, and the velocity was modified for each participant. The body mass was measured by a scale (Mettler Toltdo ID1, Columbus, OH, USA) after the first 20 min of DHR, when each participant stopped running, took off all clothes and shoes, and wiped off sweat. This was repeated every 10 min thereafter for the same duration as that of the previous study (40–60 min) . After the body mass measurement, each participant ingested either spring water or OS-1 for the amount of the body mass decrease in the time period (Fig. 1). In the second bout, the protocol was the same as that of the first bout, thus the participants ran the same duration for the same distance at the same velocities for the two bouts.
During DHR, heart rate (HR) (Model S610i; Polar Electro Oy, Finland) and rating of perceived exertion (RPE, 6–20 point Borg Scale) were recorded (Fig. 1). Ratings of perceived thermal sensation was assessed with an 8-point thermal rating scale (0: unbearably cold to 8: unbearably hot) . These were measured before DHR, then every 5 min during DHR. Blood pressure and tympanic temperature were measured by an automatic sphygmomanometer and a digital ear thermometer (BraunThermoScan 5, USA), respectively, before, after the first 20 min and every 10 min during DHR, and at the end of DHR, when each participant stopped running and was sitting in a chair.
Approximately 8 ml of blood was drawn by a standard venepuncture from the antecubital vein before, immediately after, and 65 min after DHR (Fig. 1), while each participant was sitting on a phlebotomy chair. A portion of the blood sample (1.5 ml) was used to measure hematocrit (Hct) and hemoglobin (Hb) by a capillary method and a HemoCue (Hb 201 System, Sweden), respectively, and the plasma volume change was calculated . The normal reference range was 14–18 g/dl for Hb, and 40–54% for Hct in young adults . The rest of the blood was centrifuged for 10 min at 3000 rpm to obtain serum for the analyses of electrolyte concentrations of sodium, chlorine, potassium and magnesium, and osmolality. The electrolyte concentrations were measured by an ABBOTT Architect C160000 analyser (Abbott Park, IL, USA) using a corresponding kit for sodium, chlorine and potassium and using another kit for magnesium (Abbott Laboratories Diagnostics, Abbott Park, IL, USA). Normal reference range of serum concentration for young adults by the method was sodium: 136–146 mmol/L, potassium: 3.5–4.5 mmol/L, magnesium: 0.65–1.05 mmol/L and chlorine: 96–106 mmol/L.
Data were assessed by a Shapiro-Wilk test for the normality and a Levene test for the homogeneity of variance assumption. Two-way repeated measures of analysis of variance (ANOVA) was used to compare between conditions (spring water vs OS-1) for the changes in TF before, immediately after, and 30 and 65 min post-DHR, changes in body mass, HR, RPE, thermal sensation, blood pressure, and tympanic temperature during DHR, and changes in Hct, Hb, serum osmolality, and serum electrolyte concentrations (sodium, potassium, magnesium, chloride) before, immediately after and 65 min after DHR. When the ANOVA showed a significant time effect and/or interaction effect, a Tukey’s post-hoc test was performed for multiple comparisons. Correlations between the changes in TF and serum electrolyte concentrations were assessed by a Pearson’s product-moment. Statistical significance was set at P < 0.05, and all data were presented as mean ± standard deviation (SD).