Methods
Experimental design
This study used a cross-over study design to compare between water and OS-1® conditions for changes in TF of electrical stimulation to induce calf muscle cramp before and after DHR in the heat that resulted in 2% loss of body mass by increased sweating, using 10 healthy young men. The two conditions (water, OS-1®) were separated by a week in a counter-balanced order (five participants: water condition first, five participants: OS-1® condition first).
OS-1® contains sodium (2970 mg/L), potassium (794 mg/L), magnesium (25 mg/L), chloride (1801 mg/L), glucose (18 300 mg/L) and others (eg, phosphorus). For the water condition, a commercially available spring water was used, which contains 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).
In addition to TF, serum sodium, potassium, magnesium and chloride concentrations were measured before, immediately after and 80 min after DHR (figure 1). Haematocrit (Hct), haemoglobin (Hb) and serum osmolality were assessed as dehydration parameters, and heart rate (HR), rate of physical exertion (RPE), thermal sensation, tympanic temperature, blood pressure and body mass were measured during DHR.
Figure 1Study design and the time course of measurements taken in the study. Before DHR, blood sample was taken to assess Hct, haemoglobin and serum osmolality, and to measure serum sodium, potassium, magnesium and chloride concentrations, and TF of electrical train stimulation was measured as an indicator of muscle cramp susceptibility. During DHR, heart rate was monitored continuously, RPE and thermal sensation were recorded every 5 min, and tympanic temperature, blood pressure and body mass were measured after the first 20 min followed by every 10 min during DHR. Immediately after DHR, blood sample was taken for the analyses shown above, and TF was measured agaiN. Then, the participants were instructed to ingest spring water or OS-1® in 10 min for the amount equivalent to the volume that they lost during DHR. TF was measured at 30 and 60 min after the water or OS-1® ingestion (50 and 80 min after DHR), and blood sample was taken at 60 min after the ingestion (80 min after DHR). DHR, downhill running; Hct, haematocrit; RPE, rate of physical exertion; TF, threshold frequency.
Participants
This study was approved by the Institutional Human Research Ethics Committee and complied with the Declaration of Helsinki. All participants signed an informed consent form and completed a medical questionnaire before participating in the study. It was confirmed that all of them had good health and fitness and did not have any pathological conditions such as peripheral neuropathy and liver cirrhosis that are known to cause muscle cramps. They did not have current or previous (in 2 years at least) lower limb injuries. 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. Using the data from our pilot study showing that TF to induce muscle cramp for normal condition (no dehydration) was 21.3±1.5 Hz, and TF decreased by 15% (3 Hz) after water intake, it was estimated that nine participants were necessary. Considering a potential dropout and estimation error, 10 participants were recruited for this study.
All participants were instructed to refrain from any strenuous exercise for 1 week before the study. They were asked to consume 600 mL of spring water at 2 hours before coming to the laboratory, and refrain from any food intake for at least 2 hours before each exercise session. The food intake before the first session was recorded by each participant, and he was asked to have the same food before the second session. The participants were requested not to change their lifestyle and diet, not to take any anti-inflammatory drugs or nutritional supplements and not to perform any strenuous exercise during the week between the sessions.
Threshold frequency
The participants were familiarised with the electrical stimulation to induce muscle cramp in a familiarisation session at 1 week before the first experimental session. Here, we checked whether muscle cramp was induced by the stimulation. Electrical train stimulation was delivered to calf muscles of the kicking (dominant) leg by using a portable electrical stimulator (Compex 2, Compex Medical, Switzerland). To deliver the stimulation, one electrode (cathode) was placed over the tibialis posterior nerve in the popliteal fossa, and the other electrode (anode) was placed at the ankle between the end of posterior soleus muscle and tibialis tendon. The locations of the electrodes were marked by a semi-permanent marker to ensure the consistent electrode placement between measures in the same day and between sessions separated by a week.
Each participant was lying prone on a massage bed, and the instep was placed on the bed, which kept the ankle joint in a plantar-flexed position. Each stimulation consisted of 0.5 s duration of rise time and 2.0 s bursts of stimuli of 300 μs duration. 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 preset level (18–60 mA). The stimulation intensity for each participant was determined in the familiarisation session, and the intensity was set for the baseline TF to be between 22 and 26 Hz. The intensity was the same for the rest of the measurements and between the sessions. Thereafter, the stimulation frequency was automatically increased by 2 Hz every 30 s until muscle cramp was induced. The muscle cramp was identified by a visibly taut muscle, pain and calf muscle contraction and also reported by the participant. As soon as muscle cramp was confirmed, the investigator provided passive dorsiflexion of the foot to release the cramp. The TF at which cramp was induced was recorded for further analysis.
The test–retest reliability of the TF to induce muscle cramp was assessed between two baseline measurements within the same day separated by 15 min and between days separated by 24 hours. The coefficient of variation was 3% for the two measures in the same day as well as the two different days, and SE of the measurement was 0.8 Hz for the same day and 0.6 Hz for the different days.
Downhill running
The participants performed two bouts of DHR (slope: 5%) in a climate chamber set at 35°C–36°C and 27%–34% relative humidity until their body mass was decreased by 2%. To increase sweating, all participants wore a sauna suit for the upper body. The running velocity was between 7.5 and 9.7 km/h depending on the participants, and the velocity was changed on the condition of each participant such that the velocity was reduced when the participant struggled to maintain the velocity. The body mass was measured by a scale (Mettler Toltdo ID1, Columbus, OH, USA), when the participants stopped after the first 20 min of DHR, took off all clothes and wiped sweat. This was repeated every 10 min thereafter until the body mass was decreased by 2% of the baseline body mass of each participant.
HR, RPE and thermal sensation were measured before, then every 5 min during DHR. A HR monitor (Model S610i; Polar Electro Oy, Finland) was used to record HR during DHR, and the 6-point to 20-point Borg Scale17 was used for RPE. Thermal sensation was assessed by a thermal sensation scale (eight-point scale ranging from unbearably cold [0] to unbearable hot [8]).18 Blood pressure and tympanic temperature were measured by an automatic sphygmomanometer and a temperature probe, respectively, before, after the first 20 min and every 10 min during DHR, and at the end of DHR.
Blood sampling and analyses
Approximately 8 mL of blood was drawn by a standard venepuncture technique from the antecubital vein before, immediately after and 80 min after DHR. Using a portion of the blood sample (1.5 mL), Hct and Hb were measured by a capillary method and a HemoCue (Hb 201 System, Sweden), respectively. The rest of the blood was centrifuged for 10 min at 3000 rpm to obtain serum to be analysed for electrolyte concentrations of sodium, chlorine, potassium and magnesium, and osmolality.
Statistical analysis
Two-way repeated measures analysis of variance (ANOVA) was used to compare the changes in the TF between conditions (water vs OS-1®) before, immediately after, and 50 and 80 min after DHR. Changes in other measures (body mass, HR, RPE, thermal sensation, blood pressure, tympanic temperature) during DHR, and changes in the blood dehydration parameters (Hct, Hb, serum osmolality), serum electrolyte concentrations (sodium, potassium, magnesium, chloride) before, immediately after and 80 min after DHR were also compared between conditions using two-way repeated measures ANOVA. When the ANOVA showed a significant time effect and/or interaction effect, a Tukey’s post-hoc test was performed for multiple comparisons. Statistical significance was set at p<0.05, and all data were presented as mean±SD.