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 Captisol research buy 20-min during exercise for all treatments. Blood analysis 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 H 89 using microhematocrit tubes (Statspin, Norwood, Doramapimod 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 however 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. Results Subjects Participant physical and training characteristics

are presented in Table 1. The amount of calories consumed and macronutrient proportions from 3 day diet records were 2519 ± 405 kcal, 51 ± 7% CHO, 28 ± 6% fat, 16 ± 3% protein and 5 ± 4% alcohol. The 24-hr diet recalls prior to each trial showed 2368 ± 730 kcal, 56 ± 5% CHO, 27 ± 5% fat, 16 ± 2% protein and 1 ± 2% alcohol. The 24-hr diets were the same for all treatments. Table 1 Subject physical characteristics Variable Age, yr 29.3 ± 7.8 Height, cm 175.5 ± 3.9 Weight, kg 72.4 ± 11.1 Body fat, % 9.2 ± 4.4 Fat-free mass, kg 65.4 ± 7.3 Fat mass, kg 7.0 ± 4.8 VO2max    1 min-1 4.2 ± 0.4  ml kg-1 min-1 58.2 ± 4.8 Training hours per week 8.0 ± 2.2 Running km per week 76.0 ± 13.5 Speed at max, km h-1 17.2 ± 1.6 Values are means ± SD for 11 men.