Background: Pulsatile blood flow mediates shear stress and cyclic strain on endothelial cells, activating endothelial nitric oxide synthase (eNOS). Phosphorylation of eNOS (peNOS) at serine 1177 (S1177) is known to be regulated by phosphorylated-Akt (pAKT) at S473 and activated intercellular cell adhesion molecule-1 (ICAM-1), a key regulatory event in endothelial nitric oxide (NO) production leading to cyclic guanosine monophosphate (cGMP)- mediated-vasorelaxation. Although the effect of cyclic stretch on peNOS is documented, the effect of frequency (simulating changes in heart rate) with the stretch intensity has not been established in endothelial cells. This study aimed to investigate the frequency effect of pulsatile stretch on endothelial protein expression. Method: ICAM-1, eNOS, protein kinase B (Akt) expression and phosphorylation and downstream products of Akt signalling such as NO and cGMP levels were measured in human umbilical vein endothelial cells (HUVECs) exposed to 5–20% cyclic stretch at 0.5 or 1 Hz for 18 hours. Protein and mRNA expression were quantified using western blot and qPCR respectively. NO and cGMP were quantified using commercial assay kits. Results are represented as mean ± SEM of % control. Results: Protein levels of eNOS increased at 1 Hz compared to 0.5 Hz (66 ± 11% vs. 211 ± 37%, p = 0.0006) and as did ICAM-1 (69 ± 5% vs. 210 ± 47%, p = 0.0079). Phosphorylated eNOS at S1177 increased with cyclic stretch in contrast to decreased phosphoralated Akt at both 0.5 Hz (55 ± 8%, p < 0.001) and 1 Hz (36 ± 5%, p < 0.0001). Similarly, at 1 Hz eNOS mRNA (204 ± 27%, p = 0.0027) and NO (167 ± 10%, p < 0.0060) increased while cGMP was not different in unstretched cells. Conclusion: These results demonstrate that pulsatile stretch at 0.5 and 1 Hz mediate differential expression of proteins related to NO production in HUVECs. In addition, activation of eNOS might not be entirely Akt dependent as pAkT is down regulated with cyclic stretch.