Tendon injuries may result in variations of its mechanical properties. The published data of the tendon stiffness of small animals, such as mouse and rat, are exclusively obtained by measuring grip-to-grip (g-t-g) displacement. Local strain concentration and relative sliding of the specimens in the clamps might significantly affect the measured tendon deformation. In the present study, the mechanical properties of the rat tibialis anterior tendon measured using the proposed tendon mark method were compared to those evaluated using the g-t-g displacement method. Five male Sprague Dawley rats ( approximately 418 g) were used in this study. For the proposed method, reference marks were made on the tendons using permanent ink. A microscope video system was customized to observe and record the tendon deformation. Pattern recognition software was developed to obtain the displacement time-histories of the reference marks. The distance between the grips was approximately 7 mm; and the distance between the reference marks used for the data processing was approximately 5 mm. The cross-section areas of the specimens were measured using a custom-made slot gauge and by applying a constant compressive stress (0.15 MPa). The tendons were clamped between two custom-made metal grips and stretched on a testing machine at a constant speed (1 mm/s) up to failure. Throughout the tests, the tendon specimens were submerged in a PBS bath at 22 degrees C. The deformation of the specimens was evaluated using the g-t-g displacement method and the proposed method. The stress/strain curves obtained by using the g-t-g displacement can be characterized by an initial toe zone, a quasi-linear zone, and a final failure stage. The stress/strain curves determined using the proposed method are quite different from those obtained using the g-t-g displacement: it has a smaller toe zone and a stress-hardening transition, over which the tendon stiffness increases dramatically with the increasing strain. The tendon stiffness measured by using the g-t-g displacement method may underestimate the actual mechanical properties of tendon by approximately 43%.