It is believed that load-induced bone interstitial fluid flow activates osteocytes, which are involved in the perception and transduction of external mechanical stimuli. Interstitial bone fluid movement induced by external loads also enhances the metabolic exchange of nutrients and waste products in the lacunar-canalicular network surrounding osteocytes. In the present work three experimental studies are presented to increase our understanding of the interstitial fluid pathway and the effect of load-induced fluid flow on bone. To clarify bone’s interstitial fluid pathway, the reported ability of the tracer ferritin to form halo-shaped labeling in the mineralized matrix around blood vessels was challenged and proven to be an artifact originated by histological processing techniques. Bone interstitial space and fluid movement was then characterized to determine the effect that conditions such as osteoporosis and external loading might provoke. Using a new staining technique to characterize bone interstitial space, we found that the lacunarcanalicular porosity of cancellous bone in the proximal tibia and the canalicular diameter of ovariectomized (OVX) rats were greater compared to control (CTRL). We also detected microstructural changes in the rat proximal tibia following OVX and found that higher mineralized bone regions had a more disconnected canalicular network, while lower mineralized bone regions had higher canalicular density. Finally, the combined effect of OVX and application of an external non-invasive mechanical load resulted in enhancement of injected tracer transported to osteocyte lacunae in the cancellous bone of the rat proximal tibia. The increased tracer movement may be related not only to the applied loading, but also to the canalicular widening occurring after the development of osteopenia due to OVX. The increased lacunar-canalicular porosity might disrupt the tethers between the osteocyte and the lacunar-canalicular wall, resulting in a reduced capability of estrogen-depleted animals to respond to mechanical stimuli. The bone microporosity measurements, the effect of ovariectomy and loading on transport through the lacunar-canalicular network, along with the ferritin findings will contribute to a better understanding of the effect of osteoporosis on bone morphology and on the efficiency of bone’s mechanosensory system