Inertial measurement unit (IMU) records of human movement can be converted into joint angles using a sensor-to-segment calibration, also called functional calibration. This study aims to compare the accuracy and reproducibility of four functional calibration procedures for the 3D tracking of the lower limb joint angles of young healthy individuals in gait. Three methods based on segment rotations and one on segment accelerations were used to compare IMU records with an optical system for their accuracy and reproducibility. The squat functional calibration movement, offering a low range of motion of the shank, provided the least accurate measurements. A comparable accuracy was obtained in other methods with a root mean square error below 3.6° and an absolute difference in amplitude below 3.4°. The reproducibility was excellent in the sagittal plane (intra-class correlation coefficient (ICC) > 0.91, standard error of measurement (SEM) < 1.1°), good to excellent in the transverse plane (ICC > 0.87, SEM < 1.1°), and good in the frontal plane (ICC > 0.63, SEM < 1.2°). The better accuracy for proximal joints in calibration movements using segment rotations was traded to distal joints in calibration movements using segment accelerations. These results encourage further applications of IMU systems in unconstrained rehabilitative contexts.