The malleus and incus in the human middle ear are linked by the incudo-malleolar joint (IMJ). The mobility of the human IMJ under physiologically relevant acoustic stimulation and its functional role in middle-ear sound transmission are still debated. In this study, spatial stapes motions were measured during acoustic stimulation (0.25-8 kHz) in six fresh human temporal bones for two conditions of the IMJ: (1) normal IMJ and (2) IMJ with experimentally-reduced mobility. Stapes velocity was measured at multiple points on the footplate using a scanning laser Doppler vibrometry (SLDV) system, and the 3D motion components were calculated under both conditions of the IMJ. The artificial reduction of the IMJ mobility was confirmed by measuring the relative motion between the malleus and the incus. The magnitudes of the piston-like motion of the stapes increased with the reduced IMJ mobility above 2 kHz. The increase was frequency dependent and was prominent from 2 to 4 kHz and at 5.5 kHz. The magnitude ratios of the rocking-like motions to the piston-like motion were similar for both IMJ conditions. The frequency-dependent change of the piston-like motion after the reduction of the IMJ mobility suggests that the IMJ is mobile under physiologically relevant levels of acoustic stimulation, especially at frequencies above 2 kHz.