Purpose <p>Standardising methods to calculate joint angles is essential to enable the reproducibility of movement analysis in cycling. This study compared three methods for determining lower limb posture on the bike across three positions on the saddle.</p> Methods <p>Fourteen non-cyclists were assessed in two laboratory visits. The first involved determining their maximum aerobic capacity which was used in the second visit to define a sub-maximal cycling exercise intensity. Lower limb kinematics were obtained and angles for the hip, knee, and ankle were calculated using three methods (6 o’clock position, minimum knee angle, and the largest lower limb extension). Two-way analysis of variance with repeated measurements was utilised for the hip, knee, and ankle data to assess the main effects from the method of measuring angles (i.e. 6 o’clock position vs. minimum knee angle vs. largest lower limb extension) and from positions on the saddle (i.e. anterior vs. reference vs posterior), followed by post-hoc analyses.</p> Results <p>The angles obtained at the 6 o’clock position were larger than those at the minimum knee angle and the largest lower limb extension for the hip, knee, and ankle joints (<i>p</i> &lt; 0.01). Knee flexion was greater at the anterior position than the posterior and the reference, with larger angles for the reference than the posterior (<i>p</i> &lt; 0.01). The ankle was more dorsiflexed at the anterior vs. posterior positions, anterior vs. reference positions, and references vs. posterior positions (<i>p</i> &lt; 0.01).</p> Conclusion <p>All three methods were sensitive to detect changes in saddle position but data should not be interchanged due to differences in angles between methods. These results indicate that body position on the bike should always be described using a consistent method.</p>