Objective: To investigate the effect of lipid-induced insulin resistance and type 2 diabetes on skeletal muscle calpain-10 mRNA and protein levels. Research design and methods: In the first part of this study, 10 healthy subjects underwent hyperinsulinemic euglycemic (4.5 mmol/l) clamps for 6 h with intravenous infusion of either saline (CON) or a 20% Intralipid emulsion (LIPID). Skeletal muscle biopsies were taken before and after 3 and 6 h of insulin infusion and analyzed for calpain-10 mRNA and protein expression. In the second part of the study, muscle samples obtained after an overnight fast in 10 long-standing, sedentary type 2 diabetes patients, 10 sedentary, weight-matched, normoglycemic controls, and 10 age-matched, endurance trained cyclists were analyzed for calpain-10 mRNA and protein content. Results: Intralipid infusion in healthy subjects reduced whole body glucose disposal by approximately 50% (P<0.001). Calpain-10 mRNA (P=0.01) but not protein content was reduced following 6 h of insulin infusion in both the CON and LIPID trials. Skeletal muscle calpain-10 mRNA and protein content did not differ between the type 2 diabetes patients and normoglycemic controls, but there was a strong trend for total calpain-10 protein to be greater in the endurance trained athletes (P=0.06). Conclusions: These data indicate that skeletal muscle calpain-10 expression is not modified by insulin resistance per se, and suggest that hyperinsulinemia and exercise training may modulate human skeletal muscle calpain-10 expression.