Tendon is frequently injured structure in sports activities. Stretching before activities has been recommended to prevent athletes from injuries. Clinical studies reported that stretching had effects to reduce passive muscle stiffness and leads to an increased range of motion. Recent biomechanical studies suggested that stretching might also temporary affect tensile property of tendon. However, the detailed information regarding optimizing this preconditioning has not been defined. The purpose of this study is to investigate time-dependent effect of preconditioning on the mechanical strength of tendon, and to determine an optimal duration of preconditioning for tendon strengthening.Forty eight Achilles tendons from euthanized Sprague–Dawley rats were used. The specimens were randomly divided into six groups. The specimens in control group were tested to failure without preconditioning. In preconditioning groups, 30, 100, 300, 600, and 1000s stretching at 2% strain were performed. The specimens were subsequently loaded to failure.Preconditioning from 30s to 600s significantly increased the ultimate failure load and strength. Ultimate failure load was significantly different between control and each of 30, 100, 300, and 600s stretching groups (P=0.033, 0.031, 0.004, and 0.029, respectively). There was no significant difference between control and 1000s stretching group. Ultimate strength was significantly different between control and each of 30, 100, 300, and 600s stretching groups (P=0.007, 0.008, <0.001, and 0.006, respectively), but not between control and 1000s stretching group.The time-dependent effect of preconditioning on the mechanical strength of tendon may be due to progressive collagen fibre recruitment and subsequent fatigues at the micro-level.