In bioreactor studies of tissue mechanobiology, characterizing changes in tissue quality is essential for understanding and predicting the response to mechanical stimuli. Unfortunately, current methods are often destructive and cannot be used at regular intervals on the same sample to characterize progression over time. Non-destructive methods such as low amplitude stress relaxation tests could be used, but then, the following dilemma comes into play: how can we accurately measure live tissue progression over time if the tissue is reacting to our measurement methods? In this study, we investigated the hypothesis that stress relaxation tests at physiological amplitudes conducted at regular intervals between stimulation periods do not modify tissue progression over time. Live, healthy tendons were subjected to mechanical stimuli inside a bioreactor for 3 days. The tendons were grouped based on the daily characterization protocol (24 or 0 stress relaxation tests) and their progression over time were compared. Stress relaxation tests at physiological amplitudes modified the tendon response to mechanical stimulation as observed through mechanical and histologic analyses. Possible solutions to eliminate or minimize the effect of stress relaxation tests are to use the mechanical stimuli to characterize tissue progression or to limit the number of stress relaxation tests.