Context: In rodents, high-fat diets increase intrahepatic lipid (IHL), but human studies are scarce. Objective: Our objective was to examine whether high-fat diets influence IHL, intramyocellular lipids (IMCL), and insulin resistance. Design: Twenty overweight men were randomly allocated to low- or high-fat groups (age, 54.0 +/- 2.3 and 56.4 +/- 2.5 yr; body mass index, 29.3 +/- 0.6 and 28.3 +/- 0.5 kg/m(2), respectively). Both groups started with a 3-wk low-fat diet [15% energy (En%) as protein, 65 En% as carbohydrates, 20 En% as fat], after which half of the subjects switched to a 3-wk isocaloric high-fat diet (15 En% protein, 30 En% carbohydrates, 55 En% fat). After 3 and 6 wk, IHL and IMCL content were assessed by (1)H magnetic resonance spectroscopy and a muscle biopsy, and insulin sensitivity was studied using a hyperinsulinemic-euglycemic clamp. An additional liver scan was performed after 1 wk in the high-fat group. Results: IHL decreased by 13% in the low-fat group and increased by 17% in high-fat group (P = 0.047). IMCL content was unaffected (P = 0.304). Insulin sensitivity was unaffected. At wk 3, IHL correlated negatively with insulin sensitivity (r = -0.584; P = 0.009, all subjects combined). Metabolic flexibility, defined as change in respiratory quotient upon insulin stimulation, was decreased after 3 wk of the high-fat diet (change in respiratory quotient was +0.02 +/- 0.02 vs. -0.05 +/- 0.1 in low-fat vs. high-fat group, P = 0.009). Basal plasma glucose increased after the high-fat diet (P = 0.038). Plasma parameters insulin, free fatty acids, high-sensitivity C-reactive protein, and liver enzymes and body weight were unaffected by diet. Conclusion: A 3-wk high-fat diet leads to IHL accumulation and a decreased metabolic flexibility, but insulin sensitivity is unaffected.