OBJECTIVE: To investigate the construct validity of walking knee stiffness as a measure to differentiate between individuals with and without knee osteoarthritis (OA) and the construct validity of walking knee stiffness as related to self-reported knee stiffness. The contributors to walking stiffness and its relationship with loading rate and adduction moment are also investigated. METHODS: Thirty-seven individuals with knee OA and 11 asymptomatic controls participated. Knee stiffness was calculated during walking as the change in knee flexion-extension moment divided by the change in knee flexion angle. Forward-stepwise regression models and Pearson's correlation coefficients were used to evaluate the relationships between variables. RESULTS: Knee stiffness in walking was significantly greater in the OA group (mean +/- SD 10.1 +/- 4.4 Nm/ degrees /kg x 100) compared with the controls (mean +/- SD 5.6 +/- 1.5 Nm/degrees/kg x 100) (P < 0.001). Knee excursion range explained 39% of the variance in walking knee stiffness (B = -0.736, P < 0.001) and knee extensor moment a further 7% (B = 6.974, P = 0.045). In the OA group, walking knee stiffness was not associated with self-reported stiffness (r = 0.029; P = 0.863). For the OA group, greater self-reported stiffness was associated with lower peak knee adduction moment (B = -0.354, P < 0.001). CONCLUSION: The construct validity of walking knee stiffness is supported. The poor correlation between walking stiffness and self-reported stiffness suggests the 2 measures evaluate different aspects of knee stiffness. Since a measure of walking stiffness is likely to provide valuable information, future research evaluating its clinical significance is merited.