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Introduction: Functional popliteal artery entrapment syndrome is a subtype of popliteal artery entrapment syndrome (PAES) without vascular disease or musculotendinous anomaly behind the knee. Symptoms are induced by popliteal artery extrinsic compression, leading to calf pain during lower limbs exercise. Non-invasive tests are still required to improve the diagnostic management of functional PAES. Exercise transcutaneous oxygen pressure (Ex-tcpO2) is of interest to provide objective arguments for the presence of regional blood flow impairment. Objectives: The aim of the study was to analyze whether Ex-tcpO2 could serve as a non-invasive technique for detecting ischemia resulting from PAES. Methods: Patients with suspected PAES were recruited between 2017 and 2020. The diagnosis was confirmed or rejected, according to the surgical decision based on our diagnosis management involving a multidisciplinary team. Each patient underwent Ex-tcpO2 with specific maneuvers. The decrease from rest of oxygen pressure (DROP) index served for the interpretation of exercise results. Results: Sixty-five legs with suspected PAES were recruited. Diagnosis was confirmed in 34 (52.3%) and rejected in 32 (47.7%). The average DROP values found in confirmed and rejected group at left leg were − 21.6 ± 15.4 mmHg and − 10.9 ± 11.1 mmHg, respectively (p for Mann–Whitney 0.004), and − 15.8 ± 11 mmHg and − 11.1 ± 7.5 mmHg, respectively, at right leg (p = 0.088). Ex-tcpO2 sensitivity and specificity were 52.9% and 78.1%, respectively. Conclusion: Ex-tcpO2 is an original non-invasive investigation for patients with claudication of doubtful arterial origin. The sensitivity and specificity are 52.9% and 78.1% in functional PAES diagnosis using 15 mmHg as threshold to detect ischemia during tiptoeing elevations. [ABSTRACT FROM AUTHOR]