Mechanical loading is critical for bone health. The absence of mechanical stimuli causes disuse-induced bone degradation and impaired mechanical competence, leading to osteoporosis. While it is known that reduced skeletal loading affects bone development via bone adaptation mechanisms, less is understood about bone's response to reduced loading in the mature skeleton. Evidence suggests that the osteocyte, bone’s mechanosensory cell, plays an important role in disuse-induced bone loss. The objective of this work was to investigate the effects of reduced skeletal loading and muscle paralysis on the osteocyte environment and fluid-mediated mechanotransduction in the fully developed skeleton. A preclinical murine model was used to mimic skeletal unloading due to muscle contraction impairment. Skeletally mature rats were injected in the right limb muscles with botulinum neurotoxin to induce unilateral muscle paralysis, muscle atrophy, and gait impairment. Bone’s multiscale structure, from the wholebone geometry to the osteocyte-scale microenvironment, was quantified using micro-CT imaging, confocal microscopy and super-resolution structured illumination microscopy. In addition, osteocyte biological activity and response to the disuse condition was assessed using immunohistochemical methods. Poroelastic finite element modelling was used to estimate the effects of microstructural changes associated with disuse on fluid flow within bone’s porosity, which is critical for the osteocyte’s mechanosensing ability and survival. High-resolution microCT images were used to create animal-specific finite element models of the intracortical microporosity, including the vascular pores and the osteocyte lacunae, and assuming the osteocyte-scale microporosity to be a fluid-saturated poroelastic material. Micro-CT imaging analysis demonstrated that the impairment of muscle contraction degrades cortical structure of the tibial metaphysis, reducing cortical thickness, increasing intracortical vascular porosity, and reducing osteocyte lacunar density after four weeks of disuse. Results of the numerical simulations revealed that the increased vascular porosity associated with muscle paralysis and skeletal unloading has a negative effect on the fluid flow around osteocytes embedded in the cortex, whereas lacunar density has no influence on the interstitial fluid velocity. Altogether, these results indicate that muscle activity and mechanical loading play an important role in preserving the morphology of the vascular porosity and osteocyte lacunar density in cortical bone, even if the genesis of the increased porosity remains unclear. Computational modelling demonstrated that these changes can affect fluid flow around osteocytes in the bone cortex, possibly enhancing bone degradation. Results of this thesis can help to understand the biophysical mechanisms that initiate bone loss due to disuse, possibly leading to more effective pharmacological therapies and prevention strategies.