Quantification of muscle moment arms is important for clinical evaluation of muscle pathology and treatment, and for estimating muscle and joint forces in musculoskeletal models. Moment arms estimated with musculoskeletal models often assume a default motion of the knee derived from measurements of passive cadaveric flexion. However, knee kinematics are unique to each person and activity. The objective of this study was to estimate moment arms of the knee muscles with in vivo subject- and activity-specific kinematics from seven healthy subjects performing seated knee extension and single-leg lunge to show changes between subjects and activities. 3D knee motion was measured with a high-speed stereo-radiography system. Moment arms of ten muscles were estimated in OpenSim by replacing the default knee motion with in vivo measurements. Estimated inter-subject moment arm variability was similar to previously reported in vitro measurements. RMS deviations up to 9.0 mm (35.2% of peak value) were observed between moment arms estimated with subject-specific knee extension and passive cadaveric motion. The degrees of freedom that most impacted inter-activity differences were superior/inferior and anterior/posterior translations. Musculoskeletal simulations used to estimate in vivo muscle forces and joint loads may provide significantly different results when subject- and activity-specific kinematics are implemented.