The purpose of the present investigation is to use surface interference EMG recorded by branched electrodes for assessment of muscle fatigue during sustained voluntary isometric contractions at different levels. Level-trigger averaging and turn/amplitude analysis have been applied. The conduction velocity (CV) of excitation was calculated from the time shift of the negative peaks of the averaged potentials (AvPs) derived from the EMG recorded by two electrodes placed along the muscle fibers. The recruitment of new motor units affects the negative amplitude (NA) of AvPs, the number of turns per second and the mean amplitude of turns in a different way depending on the level of sustained contractions. In contrast, the CV declined at all levels of sustained contractions and was the most appropriate parameter for the muscle fatigue assessment. There was a good correlation between CV decrease and torque reduction during sustained maximal efforts. The level-trigger averaging technique of the interference EMG recorded by surface branched electrodes is easy and non-invasive, thus being very convenient for routine application.