Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) microgel films are shown to allow controlled detachment of adsorbed cells via temperature stimuli. Cell response occurs on the timescale of several minutes, is reversible, and allows for harvesting of cells in a mild fashion. The fact that microgels are attached non-covalently allows using them on a broad variety of (charged) surfaces and is a major advantage as compared to approaches relying on covalent attachment of active films. In the following, the microgels’ physico-chemical parameters in the adsorbed state and their changes upon temperature variation are studied in order to gain a deeper understanding of the involved phenomena. By means of atomic force microscopy (AFM), the water content, mechanical properties, and adhesion forces of the microgel films are studied as a function of temperature. The analysis shows that these properties change drastically when crossing the critical temperature of the polymer film, which is the basis of the fast cell response upon temperature changes. Furthermore, nanoscale mechanical analysis shows that the films posses a nanoscopic gradient in mechanical properties.