Abstract 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.