Many studies investigating mechanisms of lipid-induced insulin resistance impose restrictive experimental conditions (constant insulin stimulation, constant glucose and/or fatty acid availability) designed to make interpretation of results simpler. We examined glucose uptake and markers of insulin action in muscle of high fat-fed (HFD) rats during the normal diurnal cycle of fasting and feeding. Male Wistar rats were fed a high fat diet (45% calories as fat) for 4 weeks. After 3 weeks of fat feeding rats were anesthetised, implanted with a single chronic jugular cannula and allowed to recover. One week after surgery the cannula was used to sample blood and deliver 14C-glucose and 3H-2-deoxyglucose to monitor in vivo glucose uptake and utilisation at 8am (the end of the feeding period) and 4pm (the end of the fasting period). Tissues were collected for further analysis. Plasma insulin levels were not different between HFD-fed and chow fed rats at either 8am or 4pm. Whole body glucose disappearance at 8am was significantly lower in HFD rats (Chow=25.2±3.4, HFD=15.3±0.3 mg/kg/min p<0.01, mean±SEM). Glucose uptake into skeletal muscle at 8am was also impaired in tibialis anterior (TA) muscle of HFD rats (Chow=6.45±0.99, HFD=4.49±0.59, p<0.05). There was no difference in the phosphorylation state of canonical signalling proteins, AKT and GSK3ß in TA muscle from chow and HFD fed rats at either time point studied. In contrast, pyruvate dehydrogenase (PDH) activity in the TA was dramatically lower in HFD rats sampled at 8am (Chow=5.17±1.46 HFD=1.05±0.18 mU/g p<0.01). These data suggest that differences in glucose uptake in muscle of HFD fed rats assessed during the normal diurnal cycle of energy metabolism relate more to changes in PDH activity than altered insulin signalling.