Tourniquet use during total knee arthroplasty (TKA) remains controversial. There are limited data demonstrating the effect of tourniquet use on flexion and extension gaps. The use of a tourniquet can theoretically affect the kinematics of the knee joint, specifically the extension and flexion gaps and the laxity, by mechanically compressing the soft tissues including the muscles above the knee joint. Therefore, this study was designed to prospectively evaluate changes in flexion and extension gaps with and without the use of a tourniquet. The following prospective study included 50 consecutive patients who underwent TKA using a surgical robot. The inclusion criteria were advanced osteoarthritis (OA) and varus-alignment or valgus-alignment <3° (hip-knee-ankle angle, standing long-leg X-ray), and the exclusion criteria were BMI >35 kg/m2 and mechanical axis in >3° valgus. A CR-TKA was performed, and the medial and lateral gaps (in mm) throughout the full range of motion in 10° increments were recorded. The procedure was conducted both with and without an applied tourniquet (350 mmHg). No significant differences were observed in the medial joint space. By contrast, the lateral gap showed significant differences in 10-20° of flexion (with a tourniquet 1.9 mm vs. without a tourniquet 2.1 mm, p = 0.018), 20-30° (1.6 vs. 1.8 mm, p = 0.02), 100-110° (0.9 vs. 1.1 mm, p = 0.021), and 110-120° (0.8 vs. 1 mm, p = 0.038). Thus, at the above degrees of flexion on the lateral side, there was a decrease in the mean of 0.2 mm with the use of a tourniquet. Although the use of a tourniquet showed a detectable change in the lateral gap in four 10° segments of flexion, clinical relevance with an average difference of 0.2 mm is not achieved. Thus, the use of a tourniquet in TKA can still be advocated based on the presented data. Level I.