Muscular capacities acting on the knee joint play an important role in many sports regarding performance improvement and injury prevention. They are often assessed by isokinetic tests assuming that the rotation centre of the knee joint remains fixed. However, sagittal knee joint movement does not occur perfectly aligned to a stable frontal axis. Nonetheless, a proper axis alignment is necessary to obtain reliable and valid results. Another important feature of highly standardised isokinetic test procedures is the subjects’ fixation. There is a controversial discussion about how subjects’ fixation affects isokinetic test findings. Most studies revealed fixation to increase force output, whereas others did not detect any differences. PURPOSE: The aim was to investigate the effects of fixation (minimal vs. maximal), contraction mode (concentric vs. eccentric) and angular velocity (30 vs. 150°/s) on the kinetics and 3D kinematics of supine knee extensions. METHODS: 18 healthy male participants (22.1y, 1.83m, 76.0kg) performed maximal unilateral isokinetic knee extensions (IsoMed 2000, D&R Ferstl GmbH, Hemau, Germany) with minimal (hand grips only) and maximal (grips, knee, hip and trunk straps) fixation. They laid in a supine position with a 20° flexed hip. At 100 Hz, two high speed cameras cameras (Basler A602fc-2, Basler AG, Ahrensburg, Germany) recorded the motion of the transepicondylar axis represented by two retro-reflective spheres (Ø 8 mm). RESULTS: Peak moments (+5%) and contractional work (+4%) significantly rose at minimal fixation. Maximal fixation improved sagittal axis alignment concerning the trajectory length of the lateral femoral epicondylus (-34%) and the mean distance to trajectory centre (-19%). Both kinematic parameters showed highly significant interactions of fixation, contraction mode and angular velocity (p<0.01). Initial axis alignment in relaxed muscular state caused an antero-cranial shift (0.8 and 2.4 cm) of the lateral femoral epicondylus as well as mean roll and yaw angle tilts of each 2.3°. CONCLUSIONS: For supine isokinetic knee extensions, hand grips suffice as fixation to obtain accurate kinematic and kinetic results. If fixation is severe, force output will decrease. To minimise misalignment, lining up should be executed when muscles are contracting isometrically.