Several laboratory studies have demonstrated a performance-enhancing effect of carbohydrate (CHO) supplementations during endurance sessions of long duration. However, the transferability of these results to real training and competition circumstances has not been conclusively shown. Therefore, we tried to test the influence of graded CHO substitution on substrate utilization and selected physiological parameters under standardized but practically orientated field conditions. Fourteen endurance-trained male subjects [mean (SD): 25 (5) years, 72 (9) kg, V˙O_2max 67 (6) ml·min^–1·kg^–1, individual anaerobic threshold (IAT) 269 (30) W] after a stepwise increasing pre-test had to perform three 4-h endurance rides on their own bicycles with simultaneous spiroergometry: constant workload 70% IAT (monitoring by SRM-System). Before and during exercise, solutions without (0%), with 6% or 12% CHO were administered double-blindly and in randomized order (total volume: 50 ml·kg^–1). After cessation of exercise, significant differences between 0% and both CHO concentrations were detected for blood glucose (GLU; 75 mg dl^–1 for 0% vs 101 mg dl^–1 for 6% vs 115 mg dl^–1 for 12%; P<0.001) and respiratory exchange ratio (0.84 vs 0.88 vs 0.90; P<0.01; correlation to GLU: r=0.46, P<0.05). Free fatty acids (0.19 vs 0.16 vs 0.10 mmol l^–1) and glycerol (0.41 vs 0.22 vs 0.12 mmol l^–1) were significantly different between the endurance trials in a dose-dependent manner (both P<0.001). Lactate concentration (P=0.42) and heart rate (P=0.12) had no significant influence from CHO substitution. We conclude that CHO substitution during 4-h endurance training inhibits lipolysis in a dose-dependent manner and enhances aerobic glycolysis. This proves that earlier laboratory findings can be replicated under field conditions using modern portable equipment.