Few studies have assessed the time-dependent response of fatigue (i.e., loss of force) during submaximal exercise without the use of maximum contractions. There is unexplored potential in the use of the superimposed muscle twitch (SIT), evoked by transcranial magnetic stimulation (TMS) or motor nerve stimulation (MNS), to assess fatigue during voluntary submaximal contractions. For the human triceps surae muscles, there are also no data on TMS-evoked twitches. To optimise the TMS stimulus for assessment of fatigue, we first tested the effects of TMS power (40, 55, 70, 85, 100% max) on SIT force during contractions (0-100% MVC in 10% increments) in six subjects. Then, we compared SIT responses (TMS and MNS) during submaximal contractions and MVCs (all at 60 s intervals) during a continuous protocol of intermittent contractions (30% MVC) consisting of consecutive 5 min periods of baseline, fatigue (ischaemia) and recovery. For TMS, SIT force increased as a diminishing function of TMS power (P < 0.05), the relationships between SIT force and the force of voluntary contraction at all TMS powers were parabolic, and SIT force was maximised at ~20-40% MVC. During intermittent contractions, MVC and SIT forces were stable during baseline, decreased similarly during ischaemia by 40-50% (P < 0.05), and recovered similarly to baseline values (P > 0.05) before the end of the protocol. TMS can be used to evoke twitches during submaximal contractions of the human calf muscle and, along with MNS, used to assess fatigue during submaximal exercise.