It is known that the mechanical response to load or displacement of a biological tissue can highly vary from one cycle to the next. Therefore, in order to establish a reference state for repeating data measurements, tissues are subjected to a preliminary step called preconditioning. Previous studies have suggested that the choice of preconditioning may influence the measured properties. To explore this effect, two different preconditioning methods were carried out on polymeric scaffolds and on bovine cartilage. The equilibrium young modulus and the energy dissipation were then measured to examine the change in the mechanical properties. Furthermore, the influence of the recovery time after preconditioning and the order of tests on the measured properties were also investigated. Results showed that the choice of preconditioning had a great impact on the measured properties as differences of more than 36% were observed for the equilibrium young modulus between samples subjected to different preconditioning protocols. The preconditioning method should therefore be informed to allow meaningful comparisons of measured data from different studies. This study also demonstrated the importance of a sufficient recovery period after preconditioning and before mechanical testing as insufficient recovery time led to irreversible damage of the samples.