Oxygen efficiency influences skeletal muscle contractile function during physiological hypoxia. Dietary fish oil, providing docosahexaenoic acid (DHA), reduces the oxygen cost of muscle contraction. This study used autologous perfused rat hindlimb model to examine the effects of a fish oil diet on skeletal muscle fatigue during an acute hypoxic challenge. Male Wistar rats were fed a diet rich in saturated fat (SF), long chain (LC) n-6 polyunsaturated fatty acids (n-6 PUFA), or LC n-3 PUFA DHA from fish oil (FO) (8weeks). During anaesthetised and ventilated conditions (normoxia 21% O2 [SaO2-98%] and hypoxia 14% O2 [SaO2-89%]) the hindlimb was perfused at a constant flow and the gastrocnemius-plantaris-soleus muscle bundle was stimulated via sciatic nerve (2Hz, 6-12V, 0.05ms) to established fatigue. Caffeine (2.5, 5, 10mM) was supplied to the contracting muscle bundle via the arterial cannula to assess force recovery. Hypoxia, independent of diet, attenuated maximal twitch tension (normoxia: 82±8; hypoxia 41±2g.g-1 tissue w.w.). However, rats fed fish oil sustained higher peak twitch tension compared to the SF and n-6 PUFA groups (P<0.05) and the time to decline to 50% of maximum twitch tension was extended (SF; 546±58, n-6PUFA; 522±58, FO; 792±96 s; P<0.05). In addition, caffeine stimulated skeletal muscle contractile recovery was enhanced in the fish oil fed animals (SF; 41±3, n-6PUFA; 40±4, FO; 52±7% recovery; P<0.05). These results support a physiological role of DHA in skeletal muscle membranes when exposed to low-oxygen stress that is consistent with the attenuation of muscle fatigue under physiologically normoxic conditions.