OBJECTIVE: To study serum levo-carnitine (l-carnitine) levels and isometric contraction, force frequency relationship and fatigue of rodent skeletal muscles in type 2 diabetes mellitus. STUDY DESIGN: Randomized controlled trial. PLACE AND DURATION OF STUDY: Physiology Department, Army Medical College, Rawalpindi, from January 2009 to January 2010. METHODOLOGY: Sixty Sprague-Dawley rats were randomly divided into two groups; group I (control), fed on normal diet ad libitum and Group II (diabetic), fed on high fat diet and administered streptozocin to induce type 2 diabetes mellitus (T2DM). At 21st day, plasma glucose and TG/HDL ratio were measured to confirm the development of T2DM in group II. At 28th day, blood was drawn by intracardiac puncture to estimate serum levo-carnitine levels. Contractile functions of skeletal muscles were assessed by using iWorx AHK/214 physiological data acquisition unit. Simple muscle twitches, maximum isometric twitch tension (MITT), time-to-peak twitch tension (TPTT) and time-to-relax to 50% of the peak twitch tension (1/2RT) of extensor digitorum muscles were recorded. Muscles were stimulated at higher frequencies to determine maximum fused tetanic tension (MFTT), maximum fused tetanic tension after fatigue protocol (TTFP) and recovery from fatigue (RF). RESULTS: Serum levo-carnitine level decreased significantly in the diabetic group. Both groups had similar MITT, TPTT and 1/2RT but decline in MFTT, TTFP and RF was significant in the diabetic rats. CONCLUSION: T2DM adversely affected serum levo-carnitine levels and the contractile functions of rodent skeletal muscle at high frequency stimulation.