Dynamization of fracture fixation is used clinically to improve the bone healing process. This study evaluated the effect of late dynamization on callus stiffness and size in a rat diaphyseal femoral osteotomy. The external unilateral fixator was dynamized by removal of the inner fixator bar, at three weeks (D3-group: n=8) or four weeks (D4-group: n=9) post-operation. Published data of a five week rigid (R-group: n=8) and flexible fixation group (F-group: n=8) were included for comparison. Preoperative and postoperative movements of the rats were measured using a motion detection system. After 5 weeks the rats were sacrificed and healing was evaluated by biomechanical and densitometric methods. By 34 days post-operation, rats from the four fixation groups had similar activity levels. There was no significant difference in flexural rigidity, callus volume or callus mineral density between the D3 and D4-groups. Both the D3-group and D4-group had significantly greater flexural rigidity (p< 0.01) and significantly lower callus total volume (p< 0.03) and callus bone volume (p< 0.03) compared to the F-group. There was no significant difference in flexural rigidity or callus mineral density between the dynamized groups compared to the R-group. However, the D3-group had less callus bone volume (p=0.06) compared to the R-group. The D4-group had significantly less callus bone volume (p=0.02) and less callus total volume (p=0.05) compared to the R-group. Late dynamization led to a stiffer callus with a smaller callus volume compared to continuously flexible fixation. The late dynamized groups had less callus volume than the continuously rigid group, but the stiffness and calcification and of the callus were similar. The late dynamized groups had undergone resorption processes, indicative of more advanced healing. Late dynamization enhanced fracture healing compared to the continuously rigid or flexible fixation.