Previous data indicates that people with type 2 diabetes rely to a greater extent on nitric oxide (NO) for skeletal muscle glucose uptake during exercise than healthy controls. Given this, in the current study we hypothesised that local NO synthase (NOS) inhibition would attenuate the increase in muscle glucose uptake during contraction in situ more in diabetic rats compared with normal chow fed rats. Sprague Dawley rats (6 weeks old, N=70) were fed a normal rat chow diet (ND) or a 22% high fat diet (HFD) for 28 days to induce insulin resistance. To induce diabetes, streptozotocin (35mg/kg) was administered at days 14 and 17 to partially compromise pancreatic function. Rats were anaesthetised and the hindlimb of one leg was stimulated (2 Hz, 0.1ms, 35V) for 30 min. The NOS inhibitor L-NAME (5µM) or saline was infused into the femoral artery of the contracting leg after 10 min. Muscle contractions increased femoral artery blood flow (~4-fold) compared to baseline. L-NAME decreased (12%, P<0.05) femoral artery blood flow compared to saline infusion without any effect on mean arterial blood pressure. Glucose uptake during contraction was significantly (P<0.05) higher in the HFD compared to ND, likely due to increased (P<0.05) plasma glucose levels in the diabetic HFD (16.9 ± 1.8 mM vs 9.7 ± 0.2 mM). L-NAME did not attenuate the increase in muscle glucose uptake during contraction in either ND (83.1 ± 5.7 vs 78.8 ± 5.4 µg/g/min) or HFD (105.2 ± 9.3 vs 110.7 ± 13.8 µg/g/min, saline vs L-NAME respectively). Despite our previous findings that NOS inhibition attenuates the increase in muscle glucose uptake during contraction in hooded Wistar rats in situ, mouse skeletal muscle ex vivo and during exercise in humans, NO does not affect glucose uptake during contraction in Sprague Dawley rats. Therefore there may be rat breed specificity affecting the role of NO on glucose uptake during contraction.