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We investigated the effects of caffeine ingestion on skeletal muscle glucose uptake, glycogen synthase (GS) activity, and insulin signaling intermediates during a 100-min euglycemic-hyperinsulinemic (100 μU/ml) clamp. On two occasions, seven men performed 1-h one-legged knee extensor exercise at 3 h before the clamp. Caffeine (5 mg/kg) or placebo was administered in a randomized, double-blind fashion 1 h before the clamp. During the clamp, whole-body glucose disposal was reduced (P < 0.05) in caffeine (37.5 ± 3.1 μmol · min⁻¹ · kg⁻¹) vs. placebo (54.1 ± 2.9 μmol · min⁻¹ · kg⁻¹). In accordance, the total area under the curve over 100 min (AUC_{0–100 min}) for insulin-stimulated glucose uptake in caffeine was reduced (P < 0.05) by ∼50% in rested and exercised muscle. Caffeine also reduced (P < 0.05) GS activity before and during insulin infusion in both legs. Exercise increased insulin sensitivity of leg …