It is well known that cyclic mechanical loading can produce an anabolic response in bone. In vivo studies have shown that the insertion of short-term recovery periods (10–15 s) into mechanical loading profiles led to an increased osteogenic response compared to continuous cyclic loading of bone. Although this is suggestive of temporal processing at the bone cell level, there is little evidence to support such a hypothesis. Therefore, the current study investigated the cellular mechanism of bone's response to rest inserted vs. continuous mechanical loading. Cell responses to rest inserted mechanical loading were quantified by applying oscillatory fluid flow (OFF) to osteoblastic cells and quantifying real-time intracellular calcium [Ca²⁺]_i, prostaglandin E₂ (PGE₂) release, and osteopontin (OPN) mRNA levels. Cells were exposed to OFF (1 Hz) at shear stresses of 1 and 2 Pa with rest periods of 5, 10, and 15 s inserted every 10 loading cycles. The insertion of 10 and 15 s rest periods into the flow profile resulted in multiple [Ca²⁺]_i responses by individual cells, increased [Ca²⁺]_i response magnitudes, and increased overall percent of cells responding compared to continuously loaded control groups. We determined the source of the multiple calcium responses to be from intracellular stores. In addition, rest inserted OFF led to similar levels of PGE₂ release and increased levels of relative OPN mRNA compared to cells exposed to continuous OFF. Our study suggests that the cellular mechanism of bone adaptation to rest inserted mechanical loading may involve modulation of intracellular levels of calcium (frequency, magnitude, percent of cells responding).