A single bout of eccentric exercise has been shown to induce muscle inflammation (Chen et al. 2003), mitochondrial swelling (Friden et al. 1983) and impair muscle glycogen storage (Asp et al. 1996) 24 to 48 hr post exercise. This study aimed to determine whether eccentric exercise could impair glucose disposal under insulin clamp conditions, and if so, whether this was associated with up-regulation of muscle pyruvate dehydrogenase kinase isoform 4 (PDK4), which phosphorylates and thereby inactivates the pyruvate dehydrogenase complex (PDC), and impaired muscle PDC activation (PDCa), a rate limiting step in carbohydrate oxidation. 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. Exercise tests were separated by at least 2 weeks and were executed in a random order. Twenty four hrs post-exercise, subjects underwent a 3 hr hyperinsulinaemic (~70 mU l-1) euglycaemic (4.5 mmol l-1) clamp to determine whole body glucose disposal, in combination with the infusion of mixed essential amino acids (18 g.hr-1; to create a ‘fed state’). Muscle biopsy samples were obtained from the vastus lateralis immediately before and after the hyperinsulinaemic clamp to determine PDCa and PDK4 protein expression levels. Statistical analyses were performed using ANOVA and a Wilcoxon non-parametric test. Data are expressed as mean±S.E.M. Whole body glucose disposal was reduced by 17.4 ± 4.9% following DR compared with FR (6.3 ± 0.5 vs 7.5 ± 0.3 mg min-1 kg-1; P<0.01). Furthermore, muscle PDK4 protein expression prior to the hyperinsulinaemic clamp was 2-fold greater following DR compared to FR (0.34 ± 0.05 vs. 0.18 ± 0.01 OD; P<0.05). Although muscle PDCa prior to the hyperinsulinaemic clamp was similar between interventions (0.27 ± 0.07 vs. 0.40 ± 0.06 mmol acetyl-CoA min-1 kg-1 wet muscle @ 37deg C, respectively; P=0.15), the increase in muscle PDCa during the clamp was blunted following DR compared with FR (Δ 0.10 ± 0.05 vs. 0.23 ± 0.08 mmol acetyl-CoA min-1 kg-1 wet muscle, respectively; P<0.01). The impairment of glucose disposal following eccentric exercise was associated with increased muscle PDK4 protein expression and inhibition of PDCa in humans under hyperinsulinaemic clamp conditions and therefore is likely to be at least partly responsible for the reported development of whole body insulin resistance. Our observations are in line with the contention that muscle inflammation contributes to the dysregulation of muscle carbohydrate metabolism via modulation of muscle PDK4 and PDC (Crossland et al. 2008).