The purpose of this study is to evaluate the changes in physical properties and biocompatibilities caused by thermocycling of CAD/CAM restorative materials (lithium disilicate, zirconia reinforced lithium silicate, polymer-infiltrated ceramic network, resin nanoceramic, highly translucent zirconia). A total of 225 specimens were prepared (12.0 × 10.0 × 1.5 mm) and divided into three groups subjected to water storage at 37 °C for 24 h (control group), 10,000 cycles in distilled water at 5–55 °C (first aged group), and 22,000 cycles in distilled water at 5–55 °C (second aged group) [(n= 15, each]). The nanoindentation hardness and Young’s modulus (nanoindenter), surface roughness (atomic force microscopy (AFM)), surface texture (scanning electron microscopy (FE-SEM)), elemental concentration (energy dispersive spectroscopy (EDS)) and contact angle were evaluated. The morphology, proliferation and adhesion of cultured human gingival fibroblasts (HGFs) were analyzed. The data were analyzed using one-way ANOVA and Tukey’s test (p < 0.05). The results showed that the nanoindentation hardness and Young’s modulus were decreased after thermocycling aging. Cell viability and proliferation of the material decreased with aging except for the highly translucent zirconia. Zirconia-reinforced lithium silicate exhibited significantly lower cell viability compared to other materials. The surface roughnesses of all groups increased with aging. Cell viability and Cell adhesion were influenced by various factors, including the surface chemical composition, hydrophilicity, surface roughness, and topography.