This study investigated the effects of prolonged exercise with and without glucose supplementation on metabolism and sarcoplasmic reticulum (SR) Ca²⁺-handling properties in working vastus lateralis muscle. Fifteen untrained volunteers (peak O₂ consumption, VO_2peak= 3.45±0.17 L/min; Mean±S.E.) cycled at ~60% VO_2peak on two occasions during which they were provided with either an artificially sweetened placebo beverage (NG) or a 6% glucose (G) beverage (~1.00 g CHO per kg body mass). Beverage supplementation started at 30 min of exercise and continued every 15 min thereafter. SR Ca²⁺-handling, metabolic and substrate responses were assessed in tissue extracted from the vastus lateralis at rest, after 30, and 90 min of exercise and at fatigue in both conditions. Plasma glucose during G was 15-23% higher (P<0.05) than those observed during NG following 60 min of exercise until fatigue. Cycle time to fatigue was increased (P<0.05) by ~19% during G (137 ± 7 min) compared to NG (115 ± 6 min). Prolonged exercise reduced (P<0.05) maximal Ca²⁺-ATPase activity (-18.4%), SR Ca²⁺-uptake (-27%), and both Phase 1 (-22.2%) and Phase 2 (-34.2%) Ca²⁺-release rates during NG. The exercise-induced reductions in SR Ca²⁺-cycling properties were not altered during G. Metabolic response to exercise were all unaltered by glucose supplementation since no differences in respiratory exchange ratios, carbohydrate and lipid oxidation rates, muscle metabolite and glycogen contents, were observed between NG and G. These results indicate the maintenance of blood glucose homeostasis by glucose supplementation is without effect in modifying the muscle metabolic, endogenous glycogen or SR Ca²⁺-handling responses.