The time course of alteration in neuromuscular function of the knee extensor muscles was characterized during a prolonged intermittent exercise. Maximal voluntary contraction (MVC) and surface EMG activity of both vastii were measured during brief interruptions before (T0), during (30, 60, 90, 120, 150 and 180 min: T30, T60, T90, T120, T150, T180) and 30 min after (T+30) a 3 h tennis match in 12 trained players. M-wave and twitch contractile properties were analyzed following single stimuli. Short tetani at 20 Hz and 80 Hz were also applied to six subjects at T0 and T180. Significant reductions in MVC (P < 0.05; -9%) and electromyographic activity normalized to the M wave for both vastii (P < 0.01) occurred with fatigue at T180. No significant changes in M-wave duration and amplitude nor in twitch contractile properties were observed. The ratio between the torques evoked by 20 Hz and 80 Hz stimulation declined significantly (P < 0.001; -12%) after exercise. Central activation failure and alterations in excitation-contraction coupling are probable mechanisms contributing to the moderate impairment of the neuromuscular function during prolonged tennis playing.