This study examined the effect of stimulation frequency (140, 200, 230 and 260 Hz) on isometric force, work loop (WL) power and the fatigue resistance of extensor digitorum longus (EDL) muscle (n=32), isolated from 8- to 10-week-old CD-1 female mice. Stimulation frequency had significant effects on isometric properties of isolated mouse EDL, whereby increasing stimulation frequency evoked increased isometric force, quicker activation and prolonged relaxation (P<0.047) up to 230 Hz and above; thereafter, force and activation did not differ (P>0.137). Increasing stimulation frequency increased maximal WL power output (P<0.001; 140 Hz, 71.3±3.5; 200 Hz, 105.4±4.1; 230 Hz, 115.5±4.1; 260 Hz, 121.1±4.1 W kg−1), but resulted in significantly quicker rates of fatigue during consecutive WLs (P<0.004). WL shapes indicate impaired muscle relaxation at the end of shortening and subsequent increased negative work appeared to contribute to fatigue at 230 and 260 Hz, but not at lower stimulation frequencies. Cumulative work was unaffected by stimulation frequency, except at the start of the fatigue protocol, where 230 and 260 Hz produced more work than 140 Hz (P<0.039). We demonstrate that stimulation frequency affects force, power and fatigue, but these effects are not uniform between different assessments of contractile performance. Therefore, future work examining the contractile properties of isolated skeletal muscle should consider increasing the stimulation frequency beyond that needed for maximal force when examining maximal power but should utilise a sub-maximal stimulation frequency for fatigue assessments to avoid a high degree of negative work atypical of in vivo function.