Atherosclerosis remains a major driver of cardiovascular disease and continues to contribute substantially to global illness and mortality. Blood flow patterns, particularly endothelial shear stress (ESS), play an important role in vascular health by influencing endothelial cell regulation of nitric oxide (NO), a molecule essential for blood vessel function and vascular homeostasis. Central to this process is the endothelial glycocalyx, including glypican-4, which acts as a mechanosensor involved in endothelial nitric oxide synthase (eNOS) activation and NO production. Previous studies have shown that glypican-4 expression increases in response to shear stress and may contribute to endothelial dysfunction under certain conditions. This study examines the relationship among shear stress, glypican-4 expression, and eNOS activation under controlled in vitro conditions that mimic exercise-induced blood flow patterns. Using Y-slides and straight slides with human umbilical vein endothelial cells (HUVECs), the study evaluates how varying flow conditions and shear stress levels affect endothelial function. Findings may deepen understanding of how exercise affects vascular adaptation and support future exercise-based approaches aimed at reducing atherosclerosis risk.