Osteoblasts respond to mechanical signals which play a key role in the formation of bone however, after extended periods of stimulation they become desensitised. Mechanosensitivity has been shown to be restored by the introduction of resting periods between loadings. The aim of this study was to analyse the effect of rest periods on the response of osteoblast-like cells seeded on collagen-glycosaminoglycan (CG) scaffolds in a flow perfusion bioreactor up to 14 days. Short (10 s) and long (7 h) term rests were incorporated into stimulation patterns. Constructs cultured in the bioreactor had a more homogenous cell distribution albeit with lower cell numbers than the static group. Osteopontin expression was significantly higher on the rest-inserted group than on the steady flow and static control. These results indicate that the insertion of short term rests during flow improves cellular distribution and osteogenic responses on CG constructs cultured in a flow perfusion bioreactor.