Aim: The aim of this report was to study the effect of high-fat (HF)/low-carbohydrate (CHO) diet on regulation of substrate metabolism in humans. Methods: Ten healthy men consumed either a HF (75% energy as fat) or control (35%) diet for 6 d in random order. On d 7, blood glucose disappearance rate (R(d)) was determined before and during a hyperinsulinemic euglycemic clamp. Substrate oxidation was determined by indirect calorimetry. Muscle biopsies were obtained prediet, postdiet, and postclamps. Results: R(d) was similar under basal conditions but slightly elevated ( approximately 10%, P < 0.05) during the last 30 min of the clamp after the HF diet. HF diet reduced CHO oxidation under basal (by approximately 40%, P < 0.05) and clamp conditions (by approximately 20%, P < 0.05), increased insulin-mediated whole-body nonoxidative glucose disposal (by 30%, P < 0.05) and muscle glycogen storage (by approximately 25%, P < 0.05). Muscle pyruvate dehydrogenase complex activity was blunted under basal and clamp conditions after HF compared with control (P < 0.05) and was accompanied by an approximately 2-fold increase (P < 0.05) in pyruvate dehydrogenase kinase 4 (PDK4) mRNA and protein expression. Conclusion: Short-term HF/low-CHO dietary intake did not induce whole-body insulin resistance, but caused a shift in im glucose metabolism from oxidation to glycogen storage. Insulin-stimulated CHO oxidation and muscle pyruvate dehydrogenase complex activity were blunted after the HF diet. Up-regulation of muscle PDK4 expression was an early molecular adaptation to these changes, and we showed for the first time in healthy humans, unlike insulin-resistant individuals, that insulin can suppress PDK4 but not PDK2 gene expression in skeletal muscle.