No comparison of the amount of low-frequency fatigue (LFF) produced by different activation frequencies exists, although frequencies ranging from 10 to 100 Hz have been used to induce LFF. The quadriceps femoris of 11 healthy subjects were tested in 5 separate sessions. In each session, the force-generating ability of the muscle was tested before and after fatigue and at 2, ∼13, and ∼38 min of recovery. Brief (6-pulse), constant-frequency trains of 9.1, 14.3, 33.3, and 100 Hz and a 6-pulse, variable-frequency train with a mean frequency of 14.3 Hz were delivered at 1 train/s to induce fatigue. Immediately postfatigue, there was a significant effect of fatiguing protocol frequency. Muscles exhibited greater LFF after stimulation with the 9.1-, 14.3-, and variable-frequency trains. These three trains also produced the greatest mean force-time integrals during the fatigue test. At 2, ∼13, and ∼38 min of recovery, however, the LFF produced was independent of the fatiguing protocol frequency. The findings are consistent with theories suggesting two independent mechanisms behind LFF and may help identify the optimal activation pattern when functional electrical stimulation is used.