Biomechanical research on tendon tissue evaluating new treatment strategies to frequently occurring clinical problems regarding tendon degeneration or trauma is of expanding scientific interest. In this context, storing tendon tissue deep-frozen is common practice to collect tissue and analyze it under equal conditions. The commonly used freezing medium, phosphate buffered saline, is known to damage cells and extracellular matrix in frozen state. Dimethyl sulfoxide, however, which is used for deep-frozen storage of cells in cell culture preserves cell vitality and reduces damage to the extracellular matrix during freezing. In our study, Achilles tendons of 26 male C57/Bl6 mice were randomized in five groups. Tendons were deep frozen in dimethyl sulfoxide or saline undergoing one or four freeze-thaw-cycles and compared to an unfrozen control group analyzing biomechanical properties, cell viability and collagenous structure. In electron microscopy, collagen fibrils of tendons frozen in saline appeared more irregular in shape, while dimethyl sulfoxide preserved the collagenous structure during freezing. In addition, treatment with dimethyl sulfoxide preserved cell viability visualized with an MTT-Assay, while tendons frozen in saline showed no remaining metabolic activity, indicating total destruction of cells during freezing. The biomechanical results revealed no differences between tendons frozen once in saline or dimethyl sulfoxide. However, tendons frozen four times in saline showed a significantly higher Young’s modulus over all strain rates compared to unfrozen tendons. In conclusion, dimethyl sulfoxide preserves the vitality of tendon resident cells and protects the collagenous superstructure during the freezing process resulting in maintained biomechanical properties of the tendon.