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Low-intensity pulsed ultrasound stimulation (LIPUS) has been proven to be a beneficial biophysical therapy for tendon-bone (T-B) healing. However, the optimal time to initiate LIPUS treatment has not been determined yet. LIPUS initiated at different stages of the inflammatory phase may profoundly affect T-B healing.An established rabbit model was used to preliminarily investigate the effect of LIPUS initiation timing on T-B healing.Controlled laboratory study.A total of 112 mature rabbits that underwent partial patellectomy were randomly assigned to 4 groups: daily mock sonication (control group) and daily ultrasonication started immediately postoperatively (immediate group), on postoperative day 7 (7-day delayed group), or on postoperative day 14 (14-day delayed group). Peripheral leukocyte counts at the inflammatory phase were used to assess postoperative inflammation. The rabbits were sacrificed at 8 or 16 weeks postoperatively for microarchitectural, histological, and mechanical evaluations of the patella-patellar tendon (PPT) junction.The biomechanical properties of the PPT junction were significantly improved in the LIPUS-treated groups. Significantly higher ultimate strength and stiffness were seen in the 7-day delayed group compared with the other groups at 8 weeks postoperatively (P < .05 for all). Newly formed bone expansion from the remaining patella in the ultrasonic treatment groups was significantly increased and remodeled compared with the control group. Micro-computed tomography analysis showed that the 7-day delayed group had significantly more bone volume and bone mineral content at the interface as compared with the other groups at 8 weeks postoperatively (P < .05 for all). Histologically, the ultrasonic treatment groups exhibited a significantly better PPT junction, as shown by more formation and remodeling of the fibrocartilage layer and newly formed bone. Additionally, peripheral leukocyte counts displayed a significant increase from postoperative day 1 to day 3 in the immediate group as compared with the other groups. Furthermore, postoperative hydrarthrosis was more likely in the immediate group.LIPUS started at postoperative day 7 had a more prominent effect on T-B healing compared with the other treatment regimens in this study.The findings of the study may help optimize the initiation timing of LIPUS for T-B healing.