Aim: Prolonged muscle activity impairs whole-muscle performance and function. However, little is known about the effects of prolonged muscle activity on the contractile function of human single muscle fibres. The purpose of this study was to investigate the effects of prolonged exercise and subsequent recovery on the contractile function of single muscle fibres obtained from elite athletes. Methods: Nine male triathletes (26 ± 1 years, 68 ± 1 mL O 2 min -1 kg -1, training volume 16 ± 1 h week -1) performed 4 h of cycling exercise (at 73% of HR max) followed by 24 h of recovery. Biopsies from vastus lateralis were obtained before and following 4 h exercise and following 24 h recovery. Measurements comprised maximal Ca 2+-activated specific force and Ca 2+ sensitivity of slow type I and fast type II single muscle fibres, as well as cycling peak power output. Results: Following cycling exercise, specific force was reduced to a similar extent in slow and fast fibres (-15 and -18%, respectively), while Ca 2+ sensitivity decreased in fast fibres only. Single fibre-specific force was fully restored in both fibre types after 24 h recovery. Cycling peak power output was reduced by 4-9% following cycling exercise and fully restored following recovery. Conclusion: This is the first study to demonstrate that prolonged cycling exercise transiently impairs specific force in type I and II fibres and decreases Ca 2+ sensitivity in type II fibres only, specifically in elite endurance athletes. Further, the changes in single fibre-specific force induced by exercise and recovery coincided temporally with changes in cycling peak power output.