Abstract Background Several clinical tests exist to assess knee laxity. Although these assessments are the predominant tools of diagnosis, they are subjective and rely on the experience of the clinician. The robotic knee testing (RKT) device has been developed to quantitatively and objectively measure rotational knee laxity. The purpose of this study was primarily to determine the intra-tester reliability of rotational knee laxity and slack, the amount of rotation occurring between the two turning points of the load deformation curve, measured by the RKT device and investigate the differences between female and male measurements. Methods Ninety-one healthy and moderately active volunteers took part in the study, of which twenty-five participated in the reliability study. Tibial rotation was performed using a servomotor to a torque of 6 N m, while measurements of motion in all 6° of freedom were collected. Reliability measurements were collected over 5 days at similar times of the day. Intra-class correlation coefficient (ICC) values and standard error of measurement (SEM) were determined across the load deformation curves. Linear mixed effects modelling was used to further assess the reliability of the measurement of external and internal tibial rotation using features of the curve (internal/external rotational laxity and slack). Measurements of internal/external rotational laxity and slack were compared between the sexes using the Student t test. Results Pointwise axial rotation measurements of the tibia had good reliability [ICC (2,1) 0.83–0.89], while reliability of the secondary motions ranged between poor and good [ICC (2,1) 0.31–0.89]. All SEMs were less than 0.3°. Most of the variation of the curve features were accounted for by inter-subject differences (56.2–77.8%) and showed moderate to good reliability. Comparison of the right legs of the sexes revealed that females had significantly larger amounts of internal rotation laxity (females 6.1 ± 1.3° vs males 5.6 ± 0.9°, p = 0.037), external rotation laxity (females 6.0 ± 1.6° vs males 5.0 ± 1.2°, p = 0.002) and slack (females 19.2 ± 4.2° vs males 16.6 ± 2.9°, p = 0.003). Similar results were seen within the left legs. Conclusions Overall, the RKT is a reliable and precise tool to assess the rotational laxity of the knee joint in healthy individuals. Finally, greater amounts of laxity and slack were also reported for females.