Dietary fish oil modifies skeletal muscle membrane fatty acid composition and oxygen efficiency similar to changes in the myocardium. Oxygen efficiency is a key determinant of sustained force in mammalian skeletal muscle. Therefore, in the present study, we tested the effects of a fish-oil diet on skeletal muscle fatigue under the stress of contraction using the rat in vivo autologous perfused hindlimb model. For 8 weeks, male Wistar rats were fed a diet rich in saturated fat (SF), a diet rich in n-6 PUFA or a diet rich in long-chain (LC) n-3 PUFA DHA derived from fish oil. In anaesthetised, mechanically ventilated rats, with their hindlimbs perfused with arterial blood at a constant flow, the gastrocnemius–plantaris–soleus muscle bundle was stimulated via sciatic nerve (2 Hz, 6–12 V, 0·05 ms) to contract repetitively for 30 min. Rats fed the n-3 PUFA diet developed higher maximum twitch tension than those fed the SF and n-6 PUFA diets (P< 0·05) and sustained twitch tension through more repetitions before the tension declined to 50 % of the maximum twitch tension (P< 0·05). The n-3 PUFA group used less oxygen for tension developed and produced higher venous lactate concentrations with no difference in glycogen utilisation compared with the SF and n-6 PUFA groups. These results further support that incorporation of DHA into skeletal muscle membranes increases the efficiency of oxygen use over a range of contractile force and this is expressed as a higher sustained force and prolonged time to fatigue.