Variations in blood pressure indicate changes in the condition of the whole body. Therefore blood pressure is a main parameter to give evidence on the physiological status of the vascular system. Although it is known that vascular endothelial cells react to a change in intravascular pressure by reorganization of the actin cytoskeleton, a direct physiological effect of pressure on the endothelium has not been investigated yet. As the dynamics of the submembranous actin web determines endothelial function, it is hypothesized that pressure directly influences vessel tone. To address the question whether pressure changes endothelial actin dynamics a method was established based on a modified AFM-based fluid chamber connected to a reservoir of variable height. The positioning of reservoir changes the hydrostatic head which allows a simulation of the mean aortic pressure (MAP). Due to the variable height of the reservoir, hydrostatic pressure can be increased by up to P = 140mmHg. We analyzed the acute influence of “normal” MAP of P = 100mmHg and elevated MAP of P =120mmHg on cell mechanics in comparison to an atmospheric control (P =0mmHg). Endothelial cells respond to an elevation of MAP by pressure-dependent increase in mechanical stiffness by 252 ± 61% (n=7) under the 120mmHg and by 348 ± 89% (n=7) under the 100mmHg which appears to be reversible. We conclude that the designed AFM-based fluid chamber allows measurements focusing on the effect of blood pressure on the endothelium. Acute application of elevated pressure stimulates changes in cortical elasticity and should control endothelial function.