Partitioning a daily mechanical stimulus into discrete loading bouts enhances bone formation in rat tibiae (J. Bone Mineral Res. 15(8) (2000) 1596). We hypothesized that a refractory period exists in primary rat osteoblastic cells, during which fluid-flow-induced [Ca^[Ca2+]i]_i oscillations are insensitive to additional short bouts (2 min) of fluid flow. Because the frequency of [Ca^[Ca2+]i]_i oscillations is believed to be important for regulating cellular activity and long-term fluid flow alters gene expression in bone cells, we also hypothesized that long-term (15 min) oscillating fluid flow produces multiple [Ca^[Ca2+]i]_i oscillations in osteoblastic cells. Primary osteoblastic cells from rat long bones were exposed to 2 min of oscillating fluid flow that produced shear stresses of 2 Pa at 2 Hz. After a rest period of 5, 30, 60, 300, 600, 900, 1800, or 2700 s, the cells were exposed to a second 2-min bout of flow. A 600 s rest period was required to recover the percentage of cells responding to fluid flow and a 900 s rest period was required to recover the [Ca^[Ca2+]i]_i oscillation magnitude. The magnitude and shape of the two [Ca^[Ca2+]i]_i oscillations were strikingly similar for individual cells after a 900 s rest period. During 15 min of continuous oscillating flow, some individual cells displayed between 1 and 9 oscillations subsequent to the initial [Ca^[Ca2+]i]_i oscillation. However, only 54% of the cells that responded initially displayed subsequent [Ca^[Ca2+]i]_i oscillations during long-term flow and the magnitude of the subsequent oscillations was only 28% of the initial response.