The molecular structure's reaction to a mechanical signal is soft tissue remodeling. It is unclear, therefore, how the molecular structure of soft tissues reacts to external mechanical stimuli. Although the origin of tissue remodeling is unknown, certain investigations have shown that it is dependent on the rate of mechanical stimulation. Developing physiotherapeutic criteria to enhance tissue health and wound healing requires an understanding of how molecular structure reacts to mechanical stimulation. In this work, we examined how the rate of mechanical stimulation affected the changes in the skin's molecular and nanostructure. Pig skin was cyclically stretched at three distinct frequencies (0.1, 2.0, and 5.0 Hz) in order to provide mechanical stimulation. In contrast, the molecular and nanostructure of mechanical tested and virgin tissue was investigated using atomic force microscope and Attenuated Total Reflectance Fourier Transform Infrared spectroscopy. On the other hand, attenuated total reflectance and an atomic force microscope (AFM) were used to evaluate the nano and molecular structure of both virgin and mechanically tested skin. respectively, Fourier transform infrared spectroscopy (FTIR–ATR). The change in collagen fibril d-periodicity and collagen molecular (tropocollagen, protein structure) was found to be much bigger at 0.1 Hz than at 2.0 and 5.0 Hz, indicating that the alteration in nano and molecular structure was sensitive to the rate of mechanical stimulation. The frequency of stretching and changes in the structure of the collagen protein were found to be significantly correlated negatively (r = 0.74, p = 0.041). These findings suggest that tissue remodeling may be enhanced by gradual mechanical stimulation during physical therapy.