During physiological gait, humans continuously modulate their knee stiffness, depending on the demands of the activity and the terrain. A similar functionality could be provided by modern actuators in transfemoral prosthesis. However, quantitative data on how knee stiffness is modulated during physiological gait is still missing. This is likely due to the experimental difficulties associated with identifying knee stiffness by applying perturbations during gait. It is our goal to quantify such stiffness modulation during gait without the need to apply perturbations. Therefore, we have recently presented an approach to quantify knee stiffness from kinematic, kinetic and electromyographic (EMG) measurements, and have validated it in isometric conditions. The goal of this paper is to extend this approach to non-isometric conditions by combining inverse dynamics and EMG measurements, and to quantify physiological stiffness modulation in the example of level-ground walking. We show that stiffness varies substantially throughout a gait cycle, with a stiffness of around 100 Nm/rad during swing phase, and a peak of 450 Nm/rad in stance phase. These quantitative results may be beneficial for design and control of transfemoral prostheses and orthoses that aim to restore physiological function.