Dietary fish oil modulates skeletal muscle membrane fatty acid composition. Similar changes in heart membrane composition modulate myocardial oxygen consumption and enhance mechanical performance. The rat in vivo autologous perfused hindlimb was used to investigate the influence of membrane composition on skeletal muscle function. Male Wistar rats were fed either saturated fat (SF), n-6 PUFA (linoleic acid rich) or n-3 PUFA (fish oil) diets for 8 weeks. Hindlimb skeletal muscle perfused using the animal’s own blood was stimulated via the sciatic nerve (1 Hz, 6-12 V, 0·05 ms) to contract in repeated 10 min bouts. The n-3 PUFA diet markedly increased 22 : 6n-3 DHA, total n-3 PUFA and decreased the n-6:n-3 PUFA ratio (P,0·05) in red and white skeletal muscle membranes. There was no difference in initial twitch tension but the n-3 PUFA group maintained greater twitch tension within all contraction bouts and recovered better during rest to produce greater twitch tension throughout the final contraction bout (P,0·05). Hindlimb oxygen consumption during contraction was significantly lower in the n-3 PUFA group compared with the SF group, producing a significantly higher O2 efficiency index compared with both SF and n-6 PUFA groups (P,0·05). Resting oxygen consumption was increased in recovery in the SF group (P,0·05) but did not change in the n-3 PUFA group. Membrane incorporation of n-3 PUFA DHA following fish oil feeding was associated with increased efficiency of muscle O2 consumption and promoted resistance to muscle fatigue.