Although it is well accepted that bone tissue metabolism is regulated by external mechanical loads, it remains unclear to what load-induced physical signals bone cells respond. In this study, a novel computer-controlled stretch device and parallel plate flow chamber were employed to investigate cytosolic calcium (Ca²⁺_i) mobilization in response to a range of dynamic substrate strain levels (0.1–10 percent, 1 Hz) and oscillating fluid flow (2 N/m², 1 Hz). In addition, we quantified the effect of dynamic substrate strain and oscillating fluid flow on the expression of mRNA for the bone matrix protein osteopontin (OPN). Our data demonstrate that continuum strain levels observed for routine physical activities (<0.5 percent) do not induce Ca²⁺_i responses in osteoblastic cells in vitro. However, there was a significant increase in the number of responding cells at larger strain levels. Moreover, we found no change in osteopontin mRNA level in response to 0.5 percent strain at 1 Hz. In contrast, oscillating fluid flow predicted to occur in the lacunar–canalicular system due to routine physical activities (2 N/m², 1 Hz) caused significant increases in both Ca²⁺_i and OPN mRNA. These data suggest that, relative to fluid flow, substrate deformation may play less of a role in bone cell mechanotransduction associated with bone adaptation to routine loads.