The aim of the present study was to estimate whether a single bout of exhaustive exercise influences the glycogen and triglyceride (TG) content in red and white gastrocnemius muscle and in the liver of rats with experimental type 2 diabetes. Experiments were carried out on male Wistar rats fed from 8 to 11 weeks of age with isocaloric standard or high-fat diet (HFD) with a previous injection of low-dose of streptozotocin (STZ) or vehicle at 2 days of age (I, control group; II, HFD; III, STZ; IV, STZ + HFD). Group IV (STZ + HFD) represents a model of type 2 diabetes. Basal liver glycogen was markedly lower in all the studied groups compared to controls. Glycogen concentration after exercise fell significantly in the examined tissues in all groups in comparison to basal conditions. A significant TG accumulation in examined tissues was observed in all the studied groups in comparison to controls. Exercise decreased tissue TG content in all the groups, but it remained significantly higher in the experimental groups vs. control. We conclude that in this model of type 2 diabetes, a single bout of exercise reveals defective utilization of tissue carbohydrates and lipids.