The dataset identifies birefringence optical retardations in sections of rat calcaneal tendons cultivated under in vitro culture conditions. Calcaneal tendons from male Wistar rats were isolated, cultured on glass slides (cov), and in direct contact with the surface of culture dishes (pla) for 8 and 12 days (8Dcov, 12Dcov, 8Dpla, and 12Dpla). Tendons that were not subjected to in vitro culture were used as controls (Control). These data provide basic information about the evaluation of changes in the optical anisotropic properties of the collagen bundles and allow the statistical determination of organizational and macromolecular characteristics of these structures.