Knee stiffness following joint injury or immobilization is a common clinical problem, and the rat has been used as a model for studies related to joint stiffness and limitation of motion. Knee stiffness measurements have been reported for the anesthetized rat, but it is difficult to separate the contributions of muscular and ligamentous restraints to the recorded values. in vitro testing of isolated rat knees devoid of musculature allows measurement of joint structural properties alone. In order to measure the effects of therapeutic or surgical interventions designed to alter joint stiffness, the opposite extremity is often used as a control. However, right–left stiffness differences for the normal rat knee have not been reported in the literature. If stiffness changes observed for a treatment group are within the normal right–left variation, validity of the results could be questioned. The objectives of this study were to utilize a new testing apparatus to measure right–left stiffness differences during knee extension in a population of normal rat knees and to document repeatability of the stiffness measurements on successive testing days. Moment versus rotation curves were recorded for 15 right–left pairs of normal rat knees on three consecutive days, with overnight specimen storage in a refrigerator. Each knee was subjected to ten loading–unloading cycles, with the last loading curve used for analysis. Angular rotation (AR), defined here as the change in flexion–extension angle from a specified applied joint moment, is commonly used as a measure of overall joint stiffness. For these tests, ARs were measured from the recorded test curves with a maximum applied extension moment of 100 g cm. Mean rotations for testing days 2 and 3 were 0.81–1.25 deg lower (p < 0.001) than for day 1, but were not significantly different from each other. For each testing day, mean rotations for right knees were 1.12–1.30 deg greater (p < 0.001) than left knees. These right–left stiffness differences should be considered when interpreting the results of knee treatment studies designed to alter knee stiffness when using the opposite extremity as a control.