The purpose of this study was to examine differences in muscle glycogen storage during three successive days of running or cycling. In a crossover design, seven male subjects performed two 3-d trials of either running (trial R) or cycling (trial C) for 60 min at 75% VO2max. Biopsy samples were obtained before and after each day's exercise from the gastrocnemius (trial R) or vastus lateralis (trial C) muscle. Diets in the 2 d preceding and during each trial contained 5 g carbohydrate.kg-1.d-1 and 14,475 +/- 402 kJ.d-1. Mean pre-exercise glycogen content (mmol.kg-1 wet wt.) was significantly reduced in both trials on day 3 (103.4 +/- 6.0) when compared to day 1 and day 2 (119.9 +/- 6.8 and 116.4 +/- 5.7, respectively). Day 1 glycogen reduction was significantly greater in trial C (P less than 0.03), and glycogen restorage was greater (P less than 0.02) only in trial C between the 1st and 2nd d. On day 3, spectrophotometric analysis of PAS strains showed that pre-exercise glycogen content in either muscle group was significantly (P less than 0.01) less in Type I as compared to Type II fibers. This difference in fiber glycogen storage did not appear to be attributable to muscle damage as negligible leukocyte infiltration and low blood CK levels were obtained. No difference between modes were observed for CK values throughout the trials. These data suggest that the depressed glycogen storage before the 3rd d of exercise was due to the moderate carbohydrate intake.