Knee joint instability is frequently (>65%) reported by patients with knee osteoarthritis (KOA) during daily activities such as gait. An objective functional measure to assess knee joint instability is lacking, making it difficult to evaluate therapies aiming to improve knee joint instability. Therefore, the aim of this thesis was to develop an objective functional measure of knee joint (in)stability in patients with knee osteoarthritis. A literature study (Chapter 2) was performed to establish an inventory of the objective parameters used for knee stability during gait. Limited or conflicting results inhibited us to recommend any of the reported parameters as measure of knee joint (in)stability. This was caused by differences in gait analysis protocols between the studies and a lack of a clear definition of dynamic knee joint stability. Therefore, we suggested a conceptual definition of knee joint stability: “The capacity to respond to a challenge during gait within the natural boundaries of the knee”. We postulate that a challenge (e.g., perturbation) is needed to evoke knee joint instability. The effects of different perturbations on muscle activation patterns of young healthy subjects was investigated in the study of Chapter 3. The results showed that with increasing intensity of the perturbation the muscle activations also became higher. Furthermore, different responses were observed in the muscles to different types of perturbations. The results of this study provides insights in how the muscles are activated to stabilize the knee when challenged. Furthermore, it could guide future studies in determining aberrant muscle activation in patients with knee disorders. Next, a study was performed to compare knee biomechanics and muscle activation patterns of 20 patients with self-reported knee joint instability (KOA-I) versus 20 patients with stable knees (KOA-S) and healthy control subjects during unperturbed (Chapter 4) and perturbed walking (Chapter 5). During unperturbed walking, higher knee flexion angles were observed during swing in patients with KOA-I. No differences were observed in the knee moments and muscle activation patterns. Dichotomization of the groups based on radiographic disease severity revealed higher knee flexion angles during initial contact (patients with moderate KOA-I) and terminal stance (patients with severe KOA-I). Furthermore, higher activations in the vastus medialis and rectus femoris were observed in patients with severe KOA-I. During perturbed walking, patients with KOA-I responded with higher knee flexion angles during terminal stance and swing. Knee muscle activation patterns were overall similar between the groups. Dichotomization of the groups revealed that patients with severe KOA-I responded with increased co-contraction of the thigh muscles. These results were combined in an assessment scheme to objectively determine knee joint instability in KOA patients. With the use of this objective assessment scheme 85% of the patients were correctly identified as “Instable” or “Stable” of the pre-determined groups based on self-reported instability. An inter-laboratory comparison of knee biomechanics and muscle activation patterns in patients with KOA was performed in Chapter 6. The results showed that the gait datasets could not be instantly merged between the laboratories due to inter-laboratory differences. Harmonization of gait analysis protocols was therefore recommended. A first step was undertaken in this harmonization process, which improved the inter-laboratory comparison. Merging of gait datasets enables testing of the objective assessment scheme on its validity and reliability. In conclusion, an assessment scheme was developed to objectively identify patients with knee joint instability during gait. This assessment scheme combines the gait deviations that were observed in patients with KOA-I during (un)perturbed gait. As the next step, the assessment scheme needs to be tested on its reliability, validity and sensitivity in a large longitudinal (international) study before it could be used in clinical practice.