Free fatty acid (FFA)- and obesity-induced insulin resistance has been associated with disturbed mitochondrial function. Elevated plasma FFA can impair insulin-induced increase of adenosine triphosphate synthesis and downregulate the expression of genes important in the biogenesis of mitochondria in human skeletal muscle. Whether FAs have a direct effect on intrinsic mitochondrial capacity remains to be established. Therefore, we measured ex vivo mitochondrial respiratory capacity in human skeletal muscle after exposure to hyperinsulinemia and high levels of plasma FFA. Nine healthy lean men were studied during a 6-hour hyperinsulinemic (600 pmol/L) euglycemic clamp with concomitant infusion of Intralipid (Fresensius Kabi Nederland, Den Bosch, the Netherlands) (FFA clamped at 0.5 mmol/L) or saline. Mitochondrial respiratory capacity was measured by high-resolution respirometry in permeabilized muscle fibers using an Oxygraph (OROBOROS Instruments, Innsbruck, Austria). Each participant served as his own control. Peripheral glucose uptake (rate of disappearance) was significantly lower during infusion of the lipid emulsion compared with the control saline infusion (68 mumol/kg.min [saline] vs 40 mumol/kg.min [lipid], P = .008). However, adenosine diphosphate-stimulated and maximal carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone-stimulated uncoupled respiration rates were not different in permeabilized skeletal muscle fibers after exposure to high levels of FFA compared with the control condition. We conclude that short-term elevation of FFA within the physiological range induces insulin resistance but does not affect intrinsic mitochondrial capacity in skeletal muscle in humans.