Physical exercise is a well-known modality for maintaining healthy locomotor mechanism. A detailed preclinical research on physical exercise effect on bone healing kinetics could help to improve the rehabilitation process after fracture treatment and bone remodeling. Our aim was to evaluate the effect of early post-operative exercise effect on bone microstructural changes in a rat model. Twenty Sprague Dawley male rats underwent bi-cortical 1.6 mm hole drilling in both femur diaphysis, after which (n = 10) underwent continuous treadmill training (TR) over two weeks, while the other group of rats (n = 10) was assigned to non-training (NT) control group. New bone formation labeling was performed by subcutaneous fluorochrome injections at day 5, 14 and 31. In vivo micro-computed tomography (μCT) scans were performed once a week during the 6-week post-operative period. Ten animals (five from each group) were euthanized at 3rd week while remaining animals were euthanized at 6th week. Femur samples were extracted and underwent ex vivo μCT and histological evaluation, while serum was used for evaluating alkaline phosphatase (ALP). μCT data demonstrated increased volume and surface of newly formed bone in defect area of TR group. Bone volume/Tissue volume (BV/TV) ratio and number of osteocytes showed an increase in TR group after 3-week period. Fluorochrome distances were increased between day 5 and 14 within the training group. Serum ALP level increased in both groups over 3- and 6-weeks. Post-operative exercise increases the bone healing kinetics and stimulates the new bone formation during and after the training protocol has ended.