The purpose of the present study was the quantification of the co-activation patterns of the knee extensor and flexor muscles during walking at self-selected speed and cadence. To this aim, the Statistical Gait Analysis, a recent methodology providing a statistical characterization of gait, was performed on surface EMG signals from Vastus Lateralis (VL) and Medial Hamstrings (MH) in 14 healthy young adult subjects. Muscular co-contraction was assessed as the overlapping period between activation intervals of agonist and antagonist muscles. Superimpositions between VL and MH activity were detected from terminal swing to the following loading response in 100% of the considered strides. This superimposition could be intended as an actual co-contraction of VL and MH, working across the same joint, the knee. It occurs in this gait phase likely in order to assist knee extension, developing muscle tension for weight acceptance during loading response, and to control knee flexion. A further less frequent (28.9±13.6% of the strides, P<;0.001) superimposition was detected in terminal stance; this superimposition, however, should not be considered a real co-contraction, because VL and MH work on different joints. These findings have the merit to provide a novel data on the variability of the reciprocal role of VL and MH during walking, allowing a deeper insight in the physiological mechanisms that regulate the knee flexion/extension.