Atomic force microscopy (AFM) has been shown to be a suitable tool to probe biophysical properties of cells and cell fragments. We analysed biophysical alterations of human platelets by AFM using streptolysin O (SLO) as a model for pore forming proteins. Permeabilization of platelet membrane by SLO was confirmed by transmission electron and confocal microscopy. Using force volume imaging combined with FIEL analysis we were able to show dynamically the increase in the elasticity of platelets during the pore formation by SLO and could correlate the viscoelasticity to the morphology of platelets. Stabilizing the actin cytoskeleton by phalloidin resulted in partial restoration of the elasticity indicating that loss of stability in platelets by SLO is mediated by alterations of both plasma membrane and cytoskeleton.