Polyethylene glycol (PEG) is used in many applications, and in the clinical field, it is one of the main components of the latest Covid vaccines. Its wide use is justified by a relatively safe profile and few known side effects. However, little is known about the biophysical effects of PEG on cells such as, cell stiffness, dry mass, and total mass. Herein, exploiting a digital holographic microscope, an inertial picobalance, and a nanoindenter, these properties are characterized in rat embryonic fibroblast exposed to different molecular weights of PEG. Immediately after the first minutes of PEG exposure to the cells, a reduction in cell dry mass can be observed as well as a rapid fluctuation in total cell weight. Cell stiffness decreases significantly after 48 h, while no important morphological changes are observed. Here, it is revealed how dry mass and total mass are rapidly and finely regulated, highlighting how the maintenance of cell density is of primary importance in cellular activities.