Native pulmonary valve leaflets (PVL) are exposed to lower pressures compared to aortic valve leaflets. Knowledge of the biology of PVL exposed to aortic pressures is limited. Hence, the study's aim was to investigate the biological properties of PVL subjected to normal aortic pressures. Porcine PVL were exposed to mean pulsatile pressures of 30 mmHg or 100 mmHg for 48 h in vitro. Subsequently, PVL were subjected to a mean pulsatile pressure of 30 mmHg for 48 h, followed by increased pressure (100 mmHg) for additional 48 h. Leaflets were evaluated by measuring collagen, DNA and sGAG contents in pressure-exposed and control PVL. Cusp morphology and cell phenotype were examined using hematoxylin and eosin staining (H and E) and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry, respectively. PVL exposed to 30 mmHg showed no significant difference (p > 0.05) in collagen, DNA or sGAG contents compared to statically incubated PVL. However, PVL exposed to 100 mmHg showed a significant increase (p < 0.05) in both collagen and sGAG contents. Collagen content was also significantly increased (p < 0.05) in PVL exposed to varying pressures for 96 h compared to PVL exposed to 30 mmHg. The morphology of PVL exposed to cyclic pressures was comparable to that of both fresh and static leaflets, while alpha-SMA expression was decreased in PVL exposed to cyclic pressures when compared to fresh PVL. PVL have the ability to withstand elevated mechanical conditions by increasing the total collagen and sGAG content of the leaflets. The structural integrity of the PVL is unaltered by changes in extracellular matrix composition. However, pulsatile pressures on the PVL did not preserve the native cell phenotype.