Fixation is a common means of preserving cells for experimental purposes. In addition to a less difficult handling of the specimen it opens up a variety of methodical options. However, it is still uncertain whether nanomechanical properties of fixated cells directly reflect those of living cells. The hypothesis was tested that mechanical (stiffness) effects induced by cytoskeletal reorganization can be identified in fixated (dead) cells similar as in living cells. We altered the structure of the actin cytoskeleton of living endothelial cells by applying cytochalasin D which depolymerizes F-actin into G-actin. Then we compared the stiffness measurements of the cell cortex (cortical stiffness) obtained in living cells to measurements in fixated cells (0.1% glutaraldehyde or 4% formaldehyde). Cortical stiffness was quantified by the analysis of force-distance curves obtained by atomic force microscopy (AFM). Our experiments show that the cytochalasin D-induced softening of the cell cortex can be detected even after the cells have been fixated. Although fixated cells are about twice as stiff as living cells the cytochalasin D-induced decrease of cortical stiffness measured with and without fixation shows a linear correlation (Pearson's r = 0.97). Plasma membrane integrity appears to be negligible, since even membrane permeabilization does not affect cortical stiffness in fixated cells. We conclude that actin nanodynamics of the cell cortex can be analyzed using fixated cells. Hence cellular nanomechanical measurements are not limited to paired AFM experiments in living cells. This opens new perspectives in medical nanodiagnostics.