Fetal immobilization affects skeletal development and can lead to severe malformations. Still, how mechanical load affects embryonic bone formation is not fully elucidated. This study combines mechanobiology, image analysis and developmental biology, to investigate the structural effects of muscular loading on embryonic long bones. We present a novel approach involving a semi-automatic workflow, to study the spatial and temporal evolutions of both hard and soft tissues in 3D without any contrast agent at micrometrical resolution. Using high-resolution phase-contrast-enhanced X-ray synchrotron microtomography, we compare the humeri of Splotch-delayed embryonic mice lacking skeletal muscles with healthy littermates. The effects of skeletal muscles on bone formation was studied from the first stages of mineral deposition (Theiler Stages 23 and 24) to just before birth (Theiler Stage 27). The results show that muscle activity affects both growth plate and mineralized regions, especially during early embryonic development. When skeletal muscles were absent, there was reduced mineralization, altered tuberosity size and location, and, at early embryonic stages, decreased chondrocyte density, size and elongation compared to littermate controls. The proposed workflow enhances our understanding of mechanobiology of early bone formation and could be implemented for the study of other complex biological tissues.