This study addresses the hypothesis that callus formation, patterning, and mineralisation are impaired during the early phase of critical sized bone defect healing, and may relate to inter-fragmentary tissue strains within the bone defect area. Twenty four 12 week old Sprague Dawley rats were used for this study. They were divided into two groups defined by the femur bone defect size: (i) 1 mm resulting in normal healing (NH), and (ii) a large sized 5 mm defect resulting in critical healing (CH). Callus formation, patterning, and mineralisation kinetics in both groups were examined in the periosteal and osteotomy gap regions using a novel longitudinal study setup. Finite element analyses on µCT generated tomograms were used to determine inter-fragmentary tissue strain patterns and compared to callus formation and patterning over the course of time. Using a novel longitudinal study technique with µCT, in vivo tracking and computer simulation approaches, this study demonstrates that: (i) periosteal bone formation and patterning are significantly influenced by bone defect size as early as 2 weeks; (ii) osteotomy gap callus formation and patterning are influenced by bone defect size, and adapt towards a non-union in critical cases by deviating into a medullary formation route as early as 2 weeks after osteotomy; (iii) the new bone formation in the osteotomy gap enclosing the medullary cavity in the CH group is highly mineralised; (iv) inter-fragmentary strain patterns predicted during the very early soft callus tissue phase (less than 2 weeks) are concurrent with callus formation and patterning at later stages. In conclusion, bone defect size influences early onset of critical healing patterns.