Intramyocellular lipid (IMCL) and plasma NEFA are important skeletal muscle fuel sources. By raising blood insulin concentrations, carbohydrate ingestion inhibits lypolysis and reduces circulating NEFA. We hypothesised that differences in the postprandial glycaemic and insulin response to carbohydrates (i.e. glycaemic index; GI) could alter NEFA availability and IMCL use during subsequent exercise. Endurance-trained individuals (n 7) cycled for 90 min at 70 % V˙O_2peak and then consumed either high GI (HGI) or low GI (LGI) meals over the following 12 h. The following day after an overnight fast, the 90 min cycle was repeated. IMCL content of the vastus lateralis was quantified using magnetic resonance spectroscopy before and after exercise. Blood samples were collected at 15 min intervals throughout exercise and analysed for NEFA, glycerol, glucose, insulin, and lactate. Substrate oxidation was calculated from expired air samples. The 90 min cycle resulted in >2-fold greater reduction in IMCL in the HGI trial (3·5 (sem 1·0) mm/kg wet weight) than the LGI trial (1·6 (sem 0·3) mm/kg wet weight, P < 0·05). During exercise, NEFA availability was reduced in the HGI trial compared to the LGI trial (area under curve 2·36 (sem 0·14) mEq/l per h v. 3·14 (sem 0·28) mEq/l per h, P < 0·05 respectively). No other differences were significant. The findings suggest that HGI carbohydrates reduce NEFA availability during exercise and increase reliance on IMCL as a substrate source during moderate intensity exercise.