Background: Proximal tibiofibular joint (PTFJ) instability is uncommon, resulting from violent twisting motions of the flexed knee during sport activities. No known image variables functionally relate the degrees of PTFJ instability. Objective: This study used ultrasound imaging to quantify the discrimination threshold for PTFJ instability and relevant functional significances. Methods: Twenty patients (mean age: 42.7 (21-59) years) with chronic PTFJ instability participated in this study. Along with the Knee Injury and Osteoarthritis Outcome Score (KOOS), the tibiofibular distances of the affected and unaffected knees were estimated with ultrasonography in the four standardized conditions at 90 degrees of flexion of the hip and knee, including neutrally relaxed position, passive internal rotation, passive external rotation, and forceful flexion of the knee joint. Results: During forceful knee flexion, the absolute tibiofibular distance in the affected leg (28.1 +/- 4.0 mm) was greater than the unaffected leg (25.1 +/- 3.7 mm). Relative changes in tibiofibular distance (RTFD) were different for both legs (affected leg: 3.9 +/- 2.7 mm; unaffected leg: 1.0 +/- 1.4 mm) (P < 0.001). The area under the receiver operator characteristic curve for the RTFD was a potent predictor of PTFJ instability (84.5%) with specificity of 95% and sensitivity of 65%. Patients with RTFD greater than 2.95 mm exhibited higher scores on the KOOS subscales of pain (P = 0.043) and quality of life (P = 0.009). Conclusion: Ultrasonic measurement of the tibiofibular distance under forceful knee flexion is clinically valuable for diagnosing PTFJ instability with functional significance. (c) 2021 Elsevier B.V. All rights reserved.