Fourteen healthy competitive runners were recruited from the University of California at Davis (UC Davis) campus and local venues. Twelve subjects were needed based on a power analysis (http://hedwig.mgh.harvard.edu/sample_size/js/js_crossover_quant.html) (power = 0.8, significance = 0.05, mean difference (MD) = 0.58 min for performance time of supplement versus water in men only and SD of the MD = 0.64 min) . Three subjects quit during the study before all trials were completed for reasons unrelated to the supplementation (aversion to needles, calf strain, knee pain). Therefore, only 11 of 14 subject’s data were included in the analysis (power = 0.8). Subjects were required to have ran a marathon in <4-hr or completed two half marathons in <2-hr within the past year and run >48 km·week-1. Medical clearance and an informed consent approved by the UC Davis Institutional Review Board were also required.
Training and diet
Subjects recorded all training sessions for the week prior to the first sub-maximal exercise test and repeated that same exercise program for the remainder of the study. Subjects were advised to rest or have a light training day prior to all testing days. The subjects’ general diets were monitored by a 3-day diet record completed before the first meeting. 24-hour recalls were completed the day prior to the first sub-maximal exercise trial and repeated exactly for all subsequent trials (Food Processor SQL Version 9.2.0, ESHA Research, Salem, OR). A 240-kcal snack (68% CHO, 16% fat and 16% protein) (Clif Bar, Berkeley, CA) was provided to consume 10-hr before each of their testing times. After the provided evening snack, only water was consumed.
Maximal exercise test
Subjects reported to the laboratory for their first visit which included a medical clearance examination and maximal exercise test. Height and body mass were measured and body composition was determined via 7 sites and a Harpenden caliper . Exercise tests were performed on a treadmill (Stairmaster Clubtrack, Vancouver, WA) set at 1% incline. After a 5-min warm-up, a graded exercise test to exhaustion was completed to determine maximal oxygen consumption (VO2max). The initial speed was based on their most recent marathon pace and increased every 2-min by 0.8-km·h-1 until volitional fatigue. A metabolic cart (TrueOne 2400, ParvoMedics, Sandy, UT) was used for metabolic measurements. At the end of every 2-min stage, heart rate (HR) via a HR monitor (5410, Polar, Woodbury, NY) and rate of perceived exertion (RPE) using a 10-point scale  were measured. The treadmill speed eliciting 75%VO2max was used as the starting speed for the sub-maximal exercise trials.
Sub-maximal exercise trials
All sub-maximal trials were done 7–14 days apart. Subjects reported to the lab at ~8:15 am in a fasted state, under normal environmental conditions: 21-23 °C, 757–761 mmHg and 35-46% relative humidity. Subjects first completed the pre-exercise questionnaires: whole body muscle soreness and fatigue (marking a line on a 100 mm visual analogue scale from no pain to extreme pain or not tired to utterly exhausted) and a gastrointestinal discomfort questionnaire (GIDQ) created by our lab. The GIDQ included 7 categories (abdominal pain, heartburn, regurgitation, bloating, nausea, belching and flatulence) rated as 0 (none), 1 (mild), 2 (moderate), 3 (quite a lot), 4 (severe), 5 (very severe) and 6 (unbearable). A 22 G catheter was then inserted into a forearm vein for blood sampling. After 10-min rest, a 9-ml blood sample was obtained. A randomized nutritional treatment was given and then subjects performed the same 5-min warm up on the treadmill for all trials. This was followed by voiding and getting a pre-exercise body weight.
During the first 80-min of the first trial, the treadmill speed was adjusted to maintain 75%VO2max and the same treadmill speed increments were used for all subsequent trials. Every 20-min during the 80-min exercise bout, GI symptoms were recorded and a 9-ml blood sample was taken while the subject stopped and straddled the treadmill for ~2-min while consuming their treatment. HR, oxygen consumption (VO2), respiratory exchange ratio (RER) and RPE were measured during the 5-min prior to stoppages. Stopwatch time was paused during stoppages so subjects ran the full 80-min. Immediately after the 80-min, the subjects completed a 5-km TT where they controlled the speed. Only the total distance covered was shown to the subjects. The time to complete the TT and average RPE, GIDQ, and HR were recorded. After a 5-min active recovery, a post-exercise body weight was recorded. Immediate, 2-hr and 5-hr post-exercise questionnaires identical to the pre-exercise questionnaires were completed.
One of two CHO supplements (pre-exercise: 0.5 g CHO·kg BW-1 and every 20-min during exercise: 0.2 g CHO·kg BW-1) or water only was randomly assigned for each week. CHO supplements included: #1 - raisins, (31 g (~1/5 cup)): 100-kcal, 24 g CHO (glucose and fructose in 1:1 ratio), 1.6 g fiber, 0.8 g protein, 8 mg sodium, 238 mg potassium and #2 – Chews (Clif blocks) (3 pieces, 30 g): 100-kcal, 24 g CHO (brown rice syrup (45% maltose, 3% glucose, and 52% maltotriose) and cane juice (50% glucose and 50% fructose)), 70 mg sodium and 20 mg potassium. Fluid intake was kept constant at 7 ml·kg BW-1 pre-exercise and 2.5 ml·kg BW-1 every 20-min during exercise for all treatments.
Blood samples were collected in non-heparinized syringes. One drop (~20 μl) measured blood lactate (Lactate Pro, Arkray, Inc, Kyoto, Japan) and hematocrit was determined using microhematocrit tubes (Statspin, Norwood, MA). 9-ml of blood was aliquoted into two SST tubes and one lithium heparin tube and was centrifuged at 3000 rpm for 15-min. 100 μl from the lithium heparin tube was analyzed for plasma glucose, sodium, potassium, and creatine kinase (CK) levels in a Metlyte 8 reagent disc (Piccolo Xpress Chemistry Analyzer, Abaxis, Union City, CA). Serum from the SST tubes was used for free fatty acid (FFA) (Wako Chemicals, Richmond, VA) and glycerol (Sigma-Aldrich, St. Louis, MO) analysis via an enzymatic colorimetric assay adapted to a microtiter plate. Insulin analysis via chemiluminescent immunoassay (Siemens ADVIA Centaur, Deerfield, IL) was done by the UC Davis Medical Center’s clinical laboratory using a 1 ml sample from a SST tube. All samples were stored in a freezer at −30°C prior to analysis.
Calculations and statistical analysis
Energy derived from total CHO and fat oxidation was calculated using the following equations, based on gas exchange measures of non-protein RER:
Data are presented as means ± standard deviation (SD). We employed a within-subject two-way analysis of variance (ANOVA) for repeated measures with a Fisher’s PLSD post hoc analysis to determine significant differences (StatView software, Version 5.0.1, SAS Institute Inc., Cary, NC). Significance was set at p ≤ 0.05.