Vibration exercise (VE) has been suggested to improve muscle strength and power performance, due to enhanced neuromuscular demand. However, understanding of the most appropriate VE protocols is lacking, limiting the optimal use of VE in rehabilitation programs. In this study, the fatiguing effect of vibration at different frequencies was investigated by employing a force-modulation VE system. Twenty volunteers performed 12-s isometric contractions of the biceps brachii with a load consisting of a baseline force of 80% of their maximum voluntary contraction (MVC) and a superimposed sinusoidal force at 0 (control condition with no vibration), 20, 30, and 40 Hz. Mechanical fatigue was estimated by assessment of MVC decay after each task while myoelectric fatigue was estimated by analysis of multichannel electromyography (EMG) signals recorded during VE. EMG conduction velocity, spectral compression, power, and fractal dimension were estimated as indicators of myoelectric fatigue. Our results suggest vibration, in particular at 30 Hz, to produce a larger degree of fatigue as compared to control condition. These results motivate further research aiming at introducing VE in rehabilitation programs with improved training protocols.