The application of heat stress on a defect site during the healing process is a promising technique for early bone regeneration. The primary goal of this study was to investigate the effect of periodic heat shock on bone formation. MC3T3-E1 cells were seeded onto biphasic calcium phosphate (BCP) scaffolds, followed by periodic heating to evaluate osteogenic differentiation. Heat was applied to cells seeded onto scaffolds at 41 °C for 1 h once, twice, and four times a day for seven days and their viability, morphology, and differentiation were analyzed. BCP scaffolds with interconnected porous structures mimic bone biology for cellular studies. MTT and confocal studies have shown that heat shock significantly increased cell proliferation without any toxic effects. Compared to non-heated samples, heat shock enhanced calcium deposition and mineralization, which could be visualized by SEM observation and Alizarin red S staining. Immunostaining images showed the localization of osteogenic proteins ALP and OPN on heat-shocked cells. qRT-PCR analysis revealed the presence of more osteospecific markers, osteopontin (OPN), osteocalcin, collagen type X, and Runx2, in the heat-shocked samples than in the non-heated sample. Periodic heat shock significantly upregulated both heat shock proteins (HSP70 and HSP27) in differentiated MC3T3-E1 cells. The results of this study demonstrated that periodically heat applied especially two times a day was better approach for osteogenic differentiation. Hence, this work provides a define temperature and time schedule for the development of a clinical heating device in future for early bone regeneration during the postsurgical period.