Twelve recreationally active men (n = 7) and women (n = 5) were recruited for the investigation (mean ± standard deviation (SD): body mass = 69.8 ± 16.4 kg, body fat = 17.2 ± 7.9%, estimated TBW (ETBW) = 40.0 ± 10.1 kg). Prior to participation, all subjects were screened for sickle cell trait (AccuBase A1cTM, Diabetes Technologies, Inc, Thomasville, GA, USA) due to the risk of microvascular occlusion during physical exertion . Pregnant females were also excluded from participation due to possible risk of severe dehydration, and for this reason, a urine pregnancy test was adminstered to all female participants on the day of testing. Study participation was supervised and approved by a physician. The project was approved by the Institutional Review Boards from Syracuse University, SUNY Upstate Medical University, and the US Army Surgeon General. All participants provided their written informed consent prior to participating in the study.
Prior to experimental testing, an estimate of stable baseline euhydrated body mass (nearest hundredth of 1 kg) was determined for each subject from mean values obtained over consecutive days (9.3 ± 0.8 days). Each measurements were obtained semi-nude using a calibrated digital scale (WeighSouth WSI-600, Mettler-Toledo, Inc, Worthington, OH, USA). These measurements were conducted at the same time of day in the laboratory after voiding and prior to eating. Fat and fat-free body mass were also determined via Dual Energy X-ray Absorptiometry (DEXA) (Lunar DPX; GE Medical Systems, Waukesha, WI, USA). Using the fat and fat-free mass from DEXA, ETBW was calculated using the following equation: ETBW = 0.10 kg (DEXA fat mass) + 0.73 kg (DEXA fat − free mass) [13, 21]. During this time period, subjects also completed three to six acute, submaximal heat familiarization sessions (20 to 30 min) on a cycle ergometer in the climate chamber. Subjects performed familiarization sessions until a stable steady state heart rate was observed during submaximal exercise in the heat (data not shown) and until they felt they were comfortable with the testing protocol. Following stable body weight assessment and familiarization, subjects completed an experimental exercise in heat testing sessions under two conditions: (1) dehydration (DHYD) and (2) fluid replacement (FR).
Both DHYD and FR testing sessions were counterbalanced and began the morning (0700 hours) following an overnight fast (>8 h). On arrival to the laboratory, subjects voided their bladders for assessment urine specific gravity via a refractometer (PAL-10S, Atago, Belevue, WA, USA). Urine specific gravity was only evaluated at baseline to document that subjects were not dehydrated prior to baseline data collection. Body mass was measured using the digital scale previously described (0715 hours), and participants rested in the supine position of 30 min. Blood samples (approximately 5 ml) were obtained via arm venipuncture using Vacutainer® (BD, Franklin Lakes, NJ, USA) containing a clot activator and gel for serum separation (0745 hours). All blood samples were immediately transported to SUNY Medical University Hospital for clinical analysis of serum osmolality (Advanced® Model 330 Micro-Osmometer, Advanced Instruments, Inc., Norwood, MA, USA). Once blood samples were obtained, subjects were transported to the MRI scanner in the supine position using a gurney to avoid fluid shifts . Once correctly positioned in the MRI scanner (0800 hours), 5-mm-thick transaxial images (2,122-ms repetition time, 0.5-mm slice-to-slice interval) were acquired using a 1.5-T Phillips Intera whole body scanner with the software Release 11 (Phillips Medical Systems, Bothell, WA, USA). MRI scans were obtained along the length of the right upper leg from the head of the femur to the knee and along the right upper arm between the head of the humerus to the elbow. Once baseline data collection was completed (0900 hours), participants walked to the climate chamber to complete one of the counterbalanced exercise and heat testing sessions.
During the DHYD testing condition, participants cycled at 60 rpm on a Monark cycle ergometer in a climate chamber at 38°C, 32% humidity, and 13 km/h wind. Participants began pedaling at 1 kp but were allowed to change the cycle load if needed to achieve the desired ETBW loss. Rectal temperature (YSI 402AC, YSI Inc., Dayton, OH, USA) and heart rate (Polar A5, Polar Electro Inc., Lake Success, NY, USA) were monitored throughout the exercise sessions. During the DHYD exercise in heat session, no fluids were provided. Participants exercised in heat and were weighed every 20 to 30 min until ETBW loss reached 3% as assessed by body mass. At this time, subjects were allowed to void their bladders and walk to the resting area where a final weight was recorded. Subjects rested in the supine position until a blood sample and MRI scan was obtained 45 min and 60 min, respectively, after exercise. After dependent measures were obtained, the participants went back to the climate chamber for additional exercise in heat. No fluids were provided during acquisition of dependent measures; therefore, participants cycled in heat and were weighed until an additional 2% ETBW was lost (which equaled 5% ETBW loss total for the condition). Once 5% ETBW loss was achieved, the dependent measures (body mass, blood sample, and MRI) were obtained as described previously.
The FR condition was identical to the DHYD condition except that all fluids lost during exercise and heat were intermittently (20 to 30 min) replaced at 1.5 times the rate they were lost using a carbohydrate-electrolyte beverage (14 g carbohydrate, 200 mg sodium, 90 mg potassium/8 oz; Gatorade® Endurance, Chicago, IL,USA) . Replacement of 1.5 times the rate of fluid loss was selected in order to account for continuous sweat and urine production while dependent measures were assessed. Participants began by cycling in heat under the same intensity and thermal conditions previously described. Following 20 to 30 min of exercise in heat, the participants were removed from the thermal chamber and weighed to determine ETBW. This amount was recorded, and fluids were provided. Participants were then re-weighed with the consumed fluid and returned to the thermal chamber for an additional 20 to 30 min of exercise in heat. This process was repeated until the total amount of fluid lost after being removed from the thermal chamber totaled 3% ETBW loss. At this point, our goal was to have adequately replaced the 3%ETBW fluid, so there was no change in body mass. Participants were allowed to void their bladders and walk to the resting area where a final weight was recorded and dependent measures were assessed. Participants then returned to the heat chamber and exercised, and every 20 to 30 min, they were then weighed and given the appropriate amount of fluids. This process continued until an additional 2% ETBW was lost (then replaced); hence, the total amount of fluid lost then replaced was equivalent to 5% ETBW. At this point, the goal was to have no change in body mass from the baseline despite sweating out 5% ETBW during the exercise and heat session. After the 5% ETBW loss, time point was reached in FR, a final body mass was recorded, and dependent measures were reassessed.
Following both DHYD and FR conditions, MRI images were transferred to a computer for tracing, and skeletal muscle volume was calculated using the NIH ImageJ analysis software (NIH, Bethesda, MD, USA) . Knee extensor muscle volume was determined by adding the volumes from the rectus femoris and vasti muscles, which were analyzed between the appearances of the distal portion of the rectus femoris and the femoral neck. The number of slices analyzed across the distance of the knee extensors was subsequently divided into three sections to determine regional changes in volume. The distal region was characterized by the first third of slices (toward the knee), the mid-belly represented the middle third of slices (mid-thigh), and the proximal region comprised the upper third of slices (toward the hip). Shoulder muscle volume was determined from the sum of five to six axial slices superior to the appearance of the anterior deltoid. Elbow flexor and extensor muscles were evaluated from the combined area of the biceps brachii and triceps brachii using the sum of five to six axial slices distal to the appearance of the anterior deltoid. The same number of slices was measured for each subject at each of the testing time points, and great care was taken to ensure within-subject measurement replication. Three investigators were responsible for MRI analysis (one for each muscle/group: knee extensors, biceps/triceps, deltoid). All MRI analyzers had previously demonstrated test-retest reliability of <1% in the laboratory and were blinded to condition and time point of the images.
Urine specific gravity, chamber temperature, relative humidity, duration of chamber exposure, exercise workload, exercise heart rate, and core body temperature were compared between DHYD and FR conditions using paired t tests. Dependent measures of body mass, serum osmolality, and skeletal muscle volume were analyzed using within-subject analysis of variance (ANOVAs) with repeated measures on condition (DHYD, FR) and time (baseline, 3% ETBW loss, 5% ETBW loss). When significant condition × time interactions were determined, differences within and between conditions were evaluated using Bonferroni corrected Student's t tests. Regional (distal, mid-belly, proximal) changes in muscle volume were also evaluated post hoc using ANOVA with repeated measures on the muscle (grouped vasti muscles, rectus femoris) and time (baseline, 5% ETBW loss) with follow-up Bonferroni corrected Student's t tests. Associations among dependent variables were further explored using Pearson's correlations. MRI image quality (motion artifact) at one time point prevented the analysis of muscle volume on one male subject for the knee extensors and one female subject for the arm muscles. Therefore, both leg and arm MRI statistical analyses were performed using n = 11. Significance was determined at p < 0.05. All values are mean ± SD.