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Aims: This study aimed to analyze kinematics and kinetics of the tibiofemoral joint in healthy subjects with valgus, neutral, and varus limb alignment throughout multiple gait activities using dynamic videofluoroscopy., Methods: Five subjects with valgus, 12 with neutral, and ten with varus limb alignment were assessed during multiple complete cycles of level walking, downhill walking, and stair descent using a combination of dynamic videofluoroscopy, ground reaction force plates, and optical motion capture. Following 2D/3D registration, tibiofemoral kinematics and kinetics were compared between the three limb alignment groups., Results: No significant differences for the rotational or translational patterns between the different limb alignment groups were found for level walking, downhill walking, or stair descent. Neutral and varus aligned subjects showed a mean centre of rotation located on the medial condyle for the loaded stance phase of all three gait activities. Valgus alignment, however, resulted in a centrally located centre of rotation for level and downhill walking, but a more medial centre of rotation during stair descent. Knee adduction/abduction moments were significantly influenced by limb alignment, with an increasing knee adduction moment from valgus through neutral to varus., Conclusion: Limb alignment was not reflected in the condylar kinematics, but did significantly affect the knee adduction moment. Variations in frontal plane limb alignment seem not to be a main modulator of condylar kinematics. The presented data provide insights into the influence of anatomical parameters on tibiofemoral kinematics and kinetics towards enhancing clinical decision-making and surgical restoration of natural knee joint motion and loading., Competing Interests: This work was partially supported by the Commission for Technology and Innovation (Bern, Switzerland, Project Number 17078.1 PFLS-LS) and Medacta International SA (Castel San Pietro, Switzerland). Medacta was not involved in the data collection, nor in the analysis or the interpretation of the data. R. List and W. R. Taylor have previously received speaker’s fees from Medacta. S. F. Fucentese receives advisory fees from Medacta as part of his consultancy work, and receives consulting fees from Zimmer Biomet and Karl Storz, unrelated to this study. S. F. Fucentese is also a board member of EKA-ESSKA Osteotomy., (© 2024 Postolka et al.)