Unaccustomed eccentric exercise has been shown to disturb muscle high energy phosphates (Rodenburg et al. 1994), induce myofibre disruption (Feasson et al. 2002) and mitochondrial swelling (Fridén et al. 1983), and impair muscle glycogen storage (Asp et al. 1996) 24 to 48 h following exercise. However, contrary to what might be expected, respiration in skinned human muscle fibers in the presence of pyruvate and malate has been reported to be unchanged for up to 96 h following eccentric exercise (Walsh et al. 2001). The aim of this study was to determine the acute impact of eccentric exercise on mitochondrial ATP production rates, rather than oxygen consumption, in human muscle using a variety of mitochondrial substrates. Eight untrained healthy male volunteers (age 26.8 ± 4.1 yrs, BMI 23.4 ± 1.2 kg.m-2) performed 30 min of running at a 0% (FR) or -15% (DR) gradient on a motorized treadmill at a speed equivalent to 80% maximal oxygen uptake (VO2max) determined using an incremental gradient protocol (Feasson et al. 2002). Exercise tests were separated by at least 2 weeks and were executed in a randomized order. Twenty-four hours after each bout of exercise, a vastus lateralis biopsy sample was obtained at rest. Maximal rates of ATP production (MRAP) in freshly isolated mitochondrial suspensions were measured in the presence of a variety of substrates using a bio-luminescence technique. The maximal activities of enzyme components of the electron transport chain (ETC) were also determined. Values represent mean ± SEM and statistical comparisons between interventions were performed using a paired Student’s t-test. Maximal rates of ATP production were markedly reduced following DR compared to FR in the presence of glutamate and succinate (8.0 ± 1.3 vs. 68.8 ± 8.1 nM min-1 mg-1 mitochondrial protein; P<0.001), glutamate and malate (6.3 ± 1.4 vs. 59.5 ± 8.2 nM min-1 mg-1 mitochondrial protein; P<0.001), palmitoyl-carnitine and malate (6.4 ± 1.2 vs 23.0 ± 3.4 nM min-1 mg-1 mitochondrial protein; P<0.05), and pyruvate and malate (3.5 ± 1.0 vs. 40.3 ± 5.8 nM min-1 mg-1 mitochondrial protein; P<0.001). No difference was observed between interventions when succinate was used as a substrate, and there were no differences in activities of components of the ETC (Data not shown). Contrary to previous evidence from skinned muscle fibre preparations, it would appear that eccentric exercise is associated with a marked impairment of MRAP in humans. This may underpin at least part of the dysregulation of energy metabolism seen under these conditions.