The effect of vibration frequency during small amplitude (approximately 0.25% of muscle-tendon complex length) vibrations on muscle stiffness and phase angle of the rat gastrocnemius medialis muscle (n = 7) was investigated at four different force levels. Frequencies varying from 5 to 180 Hz were studied. Furthermore, series elastic stiffness was determined as a function of muscle force. The experiments were also simulated, using a Hill-type muscle model representing the fundamental characteristics of the experimental muscles. The frequency response found in the experiments deviated from the simulation results: stiffness and phase were lowest at about 30 Hz, whereas the simulations showed a rapid asymptotic increase of stiffness and decrease of phase angle with increasing frequency. The values levelled off at about 60 Hz. The discrepancy between experimental and simulation results is thought to be due to changes of the contractile properties of muscle, especially at low movement frequencies, where the contractile machinery has a significant influence on muscle stiffness. At frequencies of 120 Hz and above, the muscle stiffness resembled series elastic stiffness in both experimental muscles and simulations. This suggests that the contractile element contracts approximately isometrically. Functional implications of the frequency response are discussed.