Treadmills are often used in research to analyse kinematic and physiological variables. The success of transfering the results to overground running depends on the comparability of the values between the two situations. The aim of the present study was to compare the kinematics and muscle activities in overground and treadmill running. Ten male physical education students with experience in treadmill running were asked to run with a speed of 4.0 and 6.0 m/s both overground and on a Woodway treadmill. The 3D-kinematics of the limbs were studied using a two camera video tracking system. Additionally the surface EMG of six lower limb muscles and the pattern of ground contact of the right foot was registered. Both the activities of the leg muscles and several kinematic variables showed systematic changes from overground to treadmill running. On the treadmill the subjects favoured a type of running that provided them with a higher level of security. The swing amplitude of the leg, the vertical displacement and the variance in vertical and horizontal velocity were lower in treadmill running. The angle between shoe sole and ground at foot impact was also lower and the forward lean of the upper body was higher in running on the treadmill compared with the overground mode. Most of the subjects reduced their step length and increased stride frequency in treadmill running. Furthermore, the contact time in treadmill running was shorter than for overground running. The above mentioned kinematic variables were significantly different (p < 0.05). The EMG patterns of the leg muscles were generally similar between overground and treadmill modes, but some minor differences could consistently be identified.