The simultaneous effects of body composition, cardiorespiratory fitness, physical activity, and muscle fiber characteristics on lipid oxidation at basal state and during exercise were studied in a population-based group (n = 70) of middle-aged men. Body composition, oxygen uptake, and lipid oxidation were determined in a volitional maximal exercise test, physical activity with a questionnaire, muscle fiber characteristics with muscle biopsy, and resting metabolic rate and lipid oxidation at basal state with indirect calorimetry. In regression analysis, type I muscle fibers contributed significantly to lipid oxidation at basal state (r = 0.30, r2 = 0.07, P<0.05) and during low-intensity exercise (r = 0.35, r2 = 0.10, P<0.05). ANOVA revealed 7.7% (P = 0.268) lower lipid oxidation at basal state, 14% (P<0.05) lower lipid oxidation in low-intensity exercise, and 10.5% (P = 0.088) lower lipid oxidation in moderate-intensity exercise in muscle fiber tertile I (type I muscle fiber count 28.8%) compared with muscle fiber tertile III (type I muscle fiber count 71.4%). In conclusion, the muscle fiber distribution contributed significantly to lipid oxidation during low-intensity exercise in moderately active middle-aged men.