Physical exercise describes body activity which transiently increases heart rate, blood pressure and pulse pressure. The long term effect of training, in contrast, is characterized by a decrease in resting pulse rate, pulse amplitude and mean blood pressure. Since pressure controls actin dynamics and thus participates in endothelial function, we tested whether pressure per se acts as a messenger on endothelial physiology. We designed a setup that enables application of vertical (hydrostatic) pressure on living endothelial cells during simultaneous fluorescence imaging and atomic force microscopy (AFM). Hydrostatic pressure was elevated by 60 – 120 mmHg mimicking mean arterial pressure (MAP). Endothelial cells react to variations in MAP by a reversible increase in cortical stiffness indicating a rapid reorganization of the submembranous actin web. Furthermore, pulse waves were simulated by increasing the hydrostatic pressure to diastolic values (~80 mmHg), while the AFM cantilever was used as a “pressure device” that is applying “local” systolic pressure (amplitude ~40 mmHg). Within the first 15 minutes of pulse wave application, cell volume transiently decreases followed by a full recovery despite the continuation of the pressure pulses. Cell cortex stiffness reacts to pulse wave application by an immediate increase followed by a long-term gradual decrease to levels below the initial values. Summarized, endothelial cells react to vertical pressure by variation in cortical stiffness, indicating reorganization in the submembranous actin web. Vascular endothelium indeed senses and reacts to modulations of intravascular pressure. The designed AFM fluid chamber allows the characterization and quantification of the pressure effect.