The use of a treadmill to evaluate gait patterns makes it possible to analyze many gait cycles and stride to stride variations. The objective of this study was to assess the time required for a subject to habituate to walking on a treadmill. The evolution of knee kinematics and spatio-temporal parameters were analyzed to measure habituation to walking on the treadmill. To obtain this information, data were recorded on 10 healthy subjects for about 45 minutes as they walked on a treadmill. A steady state was attained for knee kinematics and most spatio-temporal parameters at the time the treadmill had attained its maximal speed (approximately 30 seconds). However, 10 minutes were necessary for stride length to become reproducible. Time for habituation to walking on a treadmill must be considered when kinematics are evaluated during gait of healthy and disabled subjects. We have shown that, at least for young, healthy individuals who are non-naïve to walking on a treadmill, a 10-minute warm-up is enough before three-dimensional knee kinematics and spatio-temporal data can be recorded.