The glycemic index (GI) of dietary carbohydrates influences glycogen storage in skeletal muscle and circulating nonesterified fatty acid (NEFA) concentrations. We hypothesized that diets differing only in GI would alter intramuscular lipid oxidation and glycogen usage in skeletal muscle and liver during subsequent exercise. Endurance-trained individuals (n = 9) cycled for 90 min at 70% Vo(2peak) and then consumed either high- or low-GI meals over the following 12 h. The following day after an overnight fast, the 90-min cycle was repeated. (1)H and (13)C magnetic resonance spectroscopy was used before and after exercise to assess intramuscular lipid and glycogen content of the vastus muscle group and liver. Blood and expired air samples were collected at 15-min intervals throughout exercise. NEFA availability was reduced during exercise in the high- compared with the low-GI trial (area under curve 44.5 +/- 6.0 vs. 38.4 +/- 7.30 mM/h, P < 0.05). Exercise elicited an approximately 55% greater reduction in intramyocellular triglyceride (IMCL) in the high- vs. low-GI trial (1.6 +/- 0.2 vs. 1.0 +/- 0.3 mmol/kg wet wt, P < 0.05). There was no difference in the exercise-induced reduction of the glycogen pool in skeletal muscle (76 +/- 8 vs. 68 +/- 5 mM) or in liver (65 +/- 8 vs. 71 +/- 4 mM) between the low- and high-GI trials, respectively. High-GI recovery diets reduce NEFA availability and increase reliance on IMCL during moderate-intensity exercise. Skeletal muscle and liver glycogen storage or usage are not affected by the GI of an acute recovery diet.