<p>The purpose of this study was to evaluate oxygen uptake kinetics and biomechanics during two and six minute walk tests in individuals with lower limb prosthesis (iLLA) and unimpaired persons. Participants performed both 2MWT and 6MWT, oxygen uptake (VO₂), heart rate (HR) and temporal-spatial parameters were recorded. Repeated measures factorial ANOVAs analyzed differences between tests and groups, Alpha was set at 0.05. There were no significant differences in VO₂ or HR between iLLA and unimpaired groups at any time point during either walk (<i>p</i> &gt; 0.05), and neither group achieved steady state during the 2MWT, whereas steady-state HR appeared after minute 4 of the 6MWT in both groups. iLLA walked significantly less distance (<i>p</i> &lt; 0.05) but had similar cadence and total steps during each test compared with unimpaired (<i>p</i> &gt; 0.05). iLLA also had significantly greater stance ratios and shorter stride length compared with the unimpaired group (<i>p</i> &lt; 0.05) and stride length was also shorter in iLLA during the 6MWT compared to the 2MWT (<i>p</i> &lt; 0.05). The 2MWT is a strong predictor of 6MWT in the unimpaired group (<i>r</i> = 0.76, <i>p</i> = 0.001) and stronger in those using prostheses (<i>r</i> = 0.94, <i>p</i> = 0.001). Although 2MWT was a strong predictor of 6MWT performance in both groups, marked physiological and biomechanical differences were observed. Our findings do not support use of 2MWT as a proxy for 6MWT in lower limb prosthesis users.</p>