Recently, inertial measurement units have been gaining popularity as a potential alternative to optical motion capture systems in the analysis of joint kinematics. In a previous study, the accuracy of knee joint angles calculated from inertial data and an extended Kalman filter and smoother algorithm was tested using ground truth data originating from a joint simulator guided by fluoroscopy-based signals. Although high levels of accuracy were achieved, the experimental setup leveraged multiple iterations of the same movement pattern and an absence of soft tissue artefacts. Here, the algorithm is tested against an optical marker-based system in a more challenging setting, with single iterations of a loaded squat cycle simulated on seven cadaveric specimens on a force-controlled knee rig. Prior to the optimisation of local coordinate systems using the REference FRame Alignment MEthod (REFRAME) to account for the effect of differences in local reference frame orientation, root-mean-square errors between the kinematic signals of the inertial and optical systems were as high as 3.8° ± 3.5° for flexion/extension, 20.4° ± 10.0° for abduction/adduction and 8.6° ± 5.7° for external/internal rotation. After REFRAME implementation, however, average root-mean-square errors decreased to 0.9° ± 0.4° and to 1.5° ± 0.7° for abduction/adduction and for external/internal rotation, respectively, with a slight increase to 4.2° ± 3.6° for flexion/extension. While these results demonstrate promising potential in the approach's ability to estimate knee joint angles during a single loaded squat cycle, they highlight the limiting effects that a reduced number of iterations and the lack of a reliable consistent reference pose inflicts on the sensor fusion algorithm's performance. They similarly stress the importance of adapting underlying assumptions and correctly tuning filter parameters to ensure satisfactory performance. More importantly, our findings emphasise the notable impact that properly aligning reference-frame orientations before comparing joint kinematics can have on results and the conclusions derived from them.