An electrically evoked twitch during a maximal voluntary contraction (twitch interpolation) is frequently used to assess central fatigue. In this study we used intact single muscle fibres to determine if intramuscular mechanisms could affect the force increase with the twitch interpolation technique. Intact single fibres from flexor digitorum brevis of NMRI mice were dissected and mounted in a chamber equipped with a force transducer. Free myoplasmic [Ca2+] ([Ca2+]i) was measured with the fluorescent Ca2+ indicator indo-1. Seven fibres were fatigued with repeated 70 Hz tetani until 40% initial force with an interpolated pulse evoked every fifth tetanus. Results showed that the force generated by the interpolated twitch increased throughout fatigue, being 9 ± 1% of tetanic force at the start and 19 ± 1% at the end (P < 0.001). This was not due to a larger increase in [Ca2+]i induced by the interpolated twitch during fatigue but rather to the fact that the force–[Ca2+]i relationship is sigmoidal and fibres entered a steeper part of the relationship during fatigue. In another set of experiments, we observed that repeated tetani evoked at 150 Hz resulted in more rapid fatigue development than at 70 Hz and there was a decrease in force (‘sag’) during contractions, which was not observed at 70 Hz. In conclusion, the extent of central fatigue is difficult to assess and it may be overestimated when using the twitch interpolation technique.