Flow perfusion bioreactors have been shown to enhance fluid transport and improve cell viability throughout tissue-engineered bone constructs. Furthermore, osteoblasts have been shown to be stimulated by flow during bioreactor culture, although the optimum flow regime to promote an osteogenic response has yet to be found. One problem is that bone cells lose their ability to respond to stimulation; however, mechanosensitivity can be restored by introducing resting periods between bouts of loading. The aim of this study was to analyze the effect of rest-insertion on the response of osteoblasts seeded on collagen-glycosaminoglycan scaffolds in a flow perfusion bioreactor over culture periods up to 14 days. Short-term rests of 5, 10, or 15 s and long-term rests of 7 h were incorporated into stimulation patterns. Cell distribution was enhanced in all flow groups, whereas static culture controls exhibited encapsulation. Cyclooxygenase-2 expression and prostaglandin E(2) levels increased significantly because of bioreactor culture over static controls. Osteopontin expression was significantly higher for the rest-inserted groups than the static control group or steady-flow group. These results indicate that the insertion of resting periods during flow enhances cellular distribution and osteogenic responses on collagen-glycosaminoglycan constructs cultured in a flow perfusion bioreactor.