Tissue engineered heart valves (TEHV) are being investigated as an alternative to current non-viable prosthetic valves and valved conduits. Studies suggest that pulse duplicator bioreactors can stimulate TEHV development. In the current study, a model system was used to determine if cyclic flexure, a major mode of heart valve deformation, has independent effects on TEHV cell and extracellular matrix (ECM) development. Ovine vascular smooth muscle cells (SMC) were seeded for 30 h onto strips of non-woven 50:50 polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) scaffold. After 4 days of incubation, SMC-seeded and unseeded scaffolds were either maintained under static conditions (static group), or subjected to unidirectional cyclic three-point flexure at a physiological frequency and amplitude in a bioreactor (flex group) for 3 weeks. After seeding or incubation, the effective stiffness (E) was measured, with SMC-seeded scaffolds further characterized by DNA, collagen, sulfated glycosaminoglycan (S-GAG), and elastin content, as well as by histology. The seeding period was over 90% efficient, with a significant accumulation of S-GAG, no significant change in E, and no collagen detected. Following 3 weeks of incubation, unseeded scaffolds exhibited no significant change in E in the flex or static groups. In contrast, E of SMC-seeded scaffolds increased 429% in the flex group (p<0.01) and 351% in the static group (p<0.01), with a trend of increased E, a 63% increase in collagen (p<0.05), increased vimentin expression, and a more homogenous transmural cell distribution in the flex versus static group. Moreover, a positive linear relationship (r2=0.996) was found between the mean E and mean collagen concentration. These results show that cyclic flexure can have independent effects on TEHV cell and ECM development, and may be useful in predicting the mechanical properties of TEHV constructed using novel scaffold materials.