Many studies have described the effects of exercise restriction on the mammalian skeleton. In particular, human and animal models have shown that reduction in weight bearing leads to generalised bone loss and deterioration of its mechanical properties. The aim of this study was to assess the effect of prolonged exercise restriction coupled with heavy calcium demands on the micro-structural, compositional and mechanical properties of the avian skeleton. The tibiae and humeri of 2-year-old laying hens housed in conventional caging (CC) and free-range (FR) housing systems were compared by mechanical testing and micro-computed tomography (microCT) scanning. Analyses of cortical, cancellous and medullary bone were performed. Mechanical testing revealed that the tibiae and humeri of birds from the FR group had superior mechanical properties relative to those of the CC group, and microCT scanning indicated larger cortical and lower medullary regions in FR group bones. Cancellous bone analysis revealed higher trabecular thickness and a higher bone volume fraction in the FR group, but no difference in mineral density. The biomechanical superiority of bones from the FR group was primarily due to structural rather than compositional differences, and this was reflected in both the cortical and cancellous components of the bones. The study demonstrated that prolonged exercise restriction in laying hens resulted in major structural and mechanical effects on the bird skeleton.