The skin experiences constant physical stimuli, such as stretching. Exposure to excessive physical stimuli stresses the skin and can accelerate aging. In this study, we applied a method that allowed human fibroblasts and keratinocytes to be perfused with media to form 3D skin equivalents that were then uniaxially 10%-stretched for 12 h per day (at either 0.01 or 0.05 Hz) for up to 7 days to form wrinkled skin-on-a-chip (WSOC). There was more wrinkling seen in skin equivalents under 0.01 Hz uniaxial stretching than there was for non-stretched skin equivalents. At 0.05 Hz, the stratum corneum almost disappeared from the skin equivalents, indicating that stretching was harmful for the epidermis. At both frequencies, the production of collagen and related proteins in the skin equivalents, such as fibronectin 10 and keratin, decreased more than those in the non-stretched equivalents, indicating that the dermis also suffered from the repeated tensile stress. These results suggest that WSOCs can be used to examine skin aging and as an in vitro tool to evaluate the efficacy of anti-wrinkle cosmetics and medicines.