Endothelial dysfunction in diabetic vascular complications involves complex interactions between metabolic disturbances, specifically oscillatory glucose (OG) and pulsatile shear stress (PSS). Although these factors have been investigated in isolation, conventional models remain limited to static high-glucose or simplified laminar flow, lacking the capacity to mimic the integrated spatiotemporal coupling found in the diabetic vasculature. By utilizing a programmable microfluidic platform, this protocol provides a valuable platform to investigate the integrated effects of synchronized oscillatory hyperglycemia and physiological PSS on endothelial cells. This protocol aims to model endothelial dysfunction under physiologically relevant coupled metabolic and mechanical conditions. The use of a polydimethylsiloxane (PDMS) chip combined with a pressure-driven control system allows for the precise, independent modulation of flow waveforms and glucose concentration profiles. The hemodynamic fidelity of the system is validated by Micro-Particle Image Velocimetry (Micro-PIV), while the cellular response is quantitatively characterized by monitoring intracellular reactive oxygen species (ROS) levels and cell viability. Representative results demonstrate that physiological PSS effectively attenuates the oxidative injury induced by OG. This effect is demonstrated by reduced intracellular ROS levels and improved cell viability under combined stimulation conditions. Depending on the research question, parameters such as shear stress patterns, glucose oscillation frequencies, and channel geometries can be adjusted. This method serves as a versatile tool for mechanistic studies of mechanobiological pathways and drug screening for therapeutic interventions in diabetic vascular disease.