Dietary fish oil (FO) modulates muscle O2 consumption and contractile function, predictive of effects on muscle fatigue. High doses unattainable through human diet and muscle stimulation parameters used engender uncertainty in their physiological relevance. We tested the hypothesis that nutritionally relevant FO doses can modulate membrane fatty acid composition and muscle fatigue. Male Sprague-Dawley rats were randomised to control (10 % olive oil (OO) by weight) or low or moderate FO diet (LowFO and ModFO) (HiDHA tuna fish oil) for 15 weeks (LowFO: 0·3 % FO, 9·7 % OO, 0·25 % energy as EPA + DHA; ModFO: 1·25 % FO, 8·75 % OO, 1·0 % energy as EPA + DHA). Hindlimb muscle function was assessed under anaesthesia in vivo using repetitive 5 s burst sciatic nerve stimulation (0·05 ms, 7–12 V, 5 Hz, 10 s duty cycle, 300 s). There were no dietary differences in maximum developed muscle force. Repetitive peak developed force fell to 50 % within 62 (SEM 10) s in controls and took longer to decline in FO-fed rats (LowFO 110 (SEM 15) s; ModFO 117 (SEM 14) s) (P<0·05). Force within bursts was better sustained with FO and maximum rates of force development and relaxation declined more slowly. The FO-fed rats incorporated higher muscle phospholipid DHA-relative percentages than controls (P<0·001). Incorporation of DHA was greater in the fast-twitch gastrocnemius (Control 9·3 (SEM 0·8) %, LowFO 19·9 (SEM 0·4), ModFO 24·3 (SEM 1·0)) than in the slow-twitch soleus muscle (Control 5·1 (SEM 0·2), LowFO 14·3 (SEM 0·7), ModFO 18·0 (SEM 1·4)) (P <0·001), which was comparable with the myocardium, in line with muscle fibre characteristics. The LowFO and ModFO diets, emulating human dietary and therapeutic supplement intake, respectively, both elicited muscle membrane DHA enrichment and fatigue resistance, providing a foundation for translating these physiological effects to humans.