Secondary bone healing is impacted by the extent of interfragmentary motion at the fracture site. It provides mechanical stimulus that is required for the formation of fracture callus. In clinical settings, interfragmentary motion is induced by physiological loading of the broken bone – for example, by weight-bearing. However, there is no consensus about when mechanical stimuli should be applied to achieve fast and robust healing response. Therefore, this study aims to identify the effect of the immediate and delayed application of mechanical stimuli on secondary bone healing.A partial tibial osteotomy was created in twelve Swiss White Alpine sheep and stabilized using an active external fixator that induced well-controlled interfragmentary motion in form of a strain gradient. Animals were randomly assigned into two groups which mimicked early (immediate group) and late (delayed group) weight-bearing. The immediate group received daily stimulation (1000 cycles/day) from the first day post-op and the delayed group from the 22nd day post-op. Healing progression was evaluated by measurements of the stiffness of the repair tissue during mechanical stimulation and by quantifying callus area on weekly radiographs. At the end of the five weeks period, callus volume was measured on the post-mortem high-resolution computer tomography (HRCT) scan.Stiffness of the repair tissue (p<0.05) and callus progression (p<0.01) on weekly radiographs were significantly larger for the immediate group compared to the delayed group. The callus volume measured on the HRCT was nearly 3.2 times larger for the immediate group than for the delayed group (p<0.01).This study demonstrates that the absence of immediate mechanical stimuli delays callus formation, and that mechanical stimulation already applied in the early post-op phase promotes bone healing.