Three-dimensional (3-D) kinematic analyses are used widely in both sport and clinical examinations.However, this procedure depends on reliable palpation of anatomical landmarks and mal-positioning of markers betweensessions may result in improperly defined segment co-ordinate system axes which will produce in-consistent jointrotations. This had led some to question the efficacy of this technique. The aim of the current investigation was to assessthe reliability of the anatomical frame definition when quantifying 3-D kinematics of the lower extremities duringrunning. Ten participants completed five successful running trials at 4.0 m·s-1 ± 5%. 3-D angular joint kinematicsparameters from the hip, knee and ankle were collected using an eight camera motion analysis system. Two staticcalibration trials were captured. The first (test) was conducted prior to the running trials following which anatomicallandmarks were removed. The second was obtained following completion of the running trials where anatomicallandmarks were re-positioned (retest). Paired samples t-tests were used to compare 3-D kinematic parameters quantifiedusing the two static trials, and intraclass correlations were employed to examine the similarities between the sagittal,coronal and transverse plane waveforms. The results indicate that no significant (p>0.05) differences were foundbetween test and retest 3-D kinematic parameters and strong (R2≥0.87) correlations were observed between test andretest waveforms. Based on the results obtained from this investigation, it appears that the anatomical co-ordinate axesof the lower extremities can be defined reliably thus confirming the efficacy of studies using this technique.