The ultrasonography contributes to investigate in vivo tendon force–strain relationship during isometric contraction. In previous studies, different methods are available to estimate the tendon strain, using different loading rates and models to fit the tendon force–strain relationship. This study was aimed to propose a standard method to characterize the in vivo tendon force–strain relationship. We investigated the influence on the force–strain relationship for medialis gastrocnemius (MG) of (1) one method which takes into account probe and joint movements to estimate the instantaneous tendon length, (2) models used to fit the force–strain relationship for uniaxial test (polynomial vs. Ogden), and (3) the loading rate on tendon strain. Subjects performed ramp-up contraction during isometric contractions at two different target speeds: 1.5 s and minimal time with ultrasound probe fixed over the muscle–tendon junction of the MG muscle. The used method requires three markers on ultrasound probe and a marker on calcaneum to take into account all movements, and was compared to the strain estimated using ultrasound images only. The method using ultrasound image only overestimated the tendon strain from 40% of maximal force. The polynomial model showed similar fitting results than the Ogden model (R 2 ¼0.98). A loading rate effect was found on tendon strain, showing a higher strain when loading rate decreases. The characterization of tendon force–strain relationship needs to be standardized by taking into account all movements to estimate tendon strain and controlling the loading rate. The polynomial model appears to be appropriate to represent the tendon force–strain relationship.