Reduced bioavailability of nitric oxide along with an increase in inflammatory cytokines, reactive oxygen species, cell adhesion molecules, and platelet hyperactivity are underlying causes of endothelial dysfunction. Bioactive peptides derived from various plants have been shown to attenuate biomarkers of endothelial dysfunction. The objective of this study was to determine the effects of hemp seed protein hydrolysates (HSPH) inmodulating biomarkers of endothelial dysfunction using an in vitro cell culture model and an ex vivo platelet activity assay. HUVEC cells were treated with HSPH from commercial variant X and ECO-commercial variant Y,followed by hydrogen peroxide to simulate oxidative stress and inflammation. Biomarkers of endothelial dysfunction were then measured to determine the protective properties of HSPH. Blood samples collected from healthy volunteers were used to determine the effect of the HSPH on ADP and hydrogen peroxide-induced platelet activity, using flow cytometry. The HSPH reduced the production of reactive oxygen species, inflammatory cytokines IL-8, IL-12p70 and IL-1β, and adhesion molecule vascular cell adhesion molecule 1. Additionally, treatment with HSPH also reduced platelet activation marker CD62P ex vivo. The results obtained from this study suggest HSPH alleviates biomarkers of endothelial dysfunction by reducing oxidative stress, inflammation and platelet activation.