The purpose of this study was to investigate the effect of freezing time on the functional behavior of mouse muscle fibers. Passive mechanical tests were performed on single soleus muscle fibers from fresh (0 month) and preserved (stored at -20°C for 3, 6, 9 and 12 months) 3 month old mice. The Young's modulus and the dynamic and the static stresses were measured. A viscoelastic Hill model of 3rd order was used to fit the experimental relaxation test data. The statistical analysis corresponding to the elastic modulus of single muscle fibers did not differ when comparing fresh and stored samples for 3 and 6 months at -20 °C. From 9 months, fibers were less resistant and the mechanical properties were damaged. The primary goal of this study was to complete the gold standard process of muscle fiber preservation for subsequent mechanical property studies. We have demonstrated that muscle fibers can be stored at -20°C for up to 6 months without altering their mechanical properties.