The effect of carbohydrate intake before and during exercise on muscle glycogen content was investigated. According to a randomized crossover study design, eight young healthy volunteers (n=8) participated in two experimental sessions with an interval of 3 weeks. In each session subjects performed 2 hours of constant-load bicycle exercise (~75% VO₂max). On one occasion (CHO) they received carbohydrates before (~150g) and during (1g^.kg^-1 bw^.hr^-1) exercise. On the other occasion they exercised after an overnight fast (F). Fiber type-specific relative glycogen content was determined by PAS staining combined with immunofluorescence in needle biopsies from m. vastus lateralis before and immediately after exercise. Pre-exercise glycogen content was higher in type IIa fibers (9.1 ± 1 x10^-2 OD/µm2) than in type I fibers (8.0 ± 1 x10^-2 OD/µm²; p<0.0001). Type IIa fiber glycogen content decreased during F from 9.6 ± 1 x10^-2 OD/µm² to 4.5 ± 1 x10^-2 OD/µm² (p=0.001), but did not significantly change during CHO (p=0.29). Conversely, in type I fibers during CHO and F the exercise bout decreased glycogen content to the same degree. We conclude that the combination of carbohydrate intake both before and during moderate to high-intensity endurance exercise results in glycogen sparing in type IIa muscle fibers.