BACKGROUND: Generalized joint hypermobility (GJH) is a highly prevalent disease that frequently affects the knee joint. The current literature has conflicting results about whether patients with GJH had knee kinematics deficiency during gait. This could be because most of the testing environment (level walking) was gentle and low-demanding for patients when studying their knee kinematics. With a high-demanding knee function and sagittal firm structure requirement, upslope walking was thought to stimulate sagittal knee kinematics deficiency in patients with GJH. RESEARCH QUESTIONS: However, only little investigation reported whether upslope walking could stimulate knee kinematic deficiency or not. We hypothesize that upslope walking can increase sagittal knee kinematic deficiency between GJH subjects and healthy controls. METHODS: A three-dimensional motion analysis was conducted to explore whether upslope walking could stimulate sagittal knee kinematic deficiency in patients with GJH. A total of 44 patients with GJH and 44 healthy controls were recruited. Subjects walked on both level and upslope (15%) conditions when the kinematic data were collected. SPM1D analysis was taken to explore the differences between groups. RESULTS: Our results showed that upslope walking could significantly increase knee flexion angle and anterior tibial translation in both GJH patients and healthy controls (p < 0.05). The increments of anterior tibial translation (values in upslope walking minus values in level walking) of GJH patients were greater than those of healthy controls (magnitude varying from 2.5 to 2.9 mm during 0-3% gait cycles (GC), p = 0.034; 1.4-2.9 mm during 93-100%GC, p = 0.012). SIGNIFICANCES: The findings partially confirmed our hypothesis and suggested that upslope walking could increase anterior tibial translation deficiency in patients with GJH. Upslope walking may be a practical motion task in studying the weakness of knee kinematics of GJH subjects for researchers and scholars. Patients with GJH may face a more challenging knee kinematic environment than healthy controls in up-sloped activities.