Objectives: Near-Infrared Spectroscopy (NIRS), suitable for dynamic measurements, is not routinely used for peripheral arterial disease (PAD). We propose a dynamic NIRS-based measurement to quantify variations in muscle metabolism in PAD. Method: Sixty-seven consecutive PAD patients (males = 56, age 71.6 ± 8.7 years) and 28 healthy subjects (males = 12, age 30.4 ± 11.9 years) were studied. An echo-colour Doppler (ECD) was performed and the ankle-brachial index (ABI) was calculated. Participants performed an incremental treadmill test with NIRS probes on the gastrocnemius. Variations in oxygenated (HbO2), deoxygenated (HHb), total (tHb = HbO2 + HHb), and differential (dHb = HbO2 - HHb) haemoglobin were recorded and quantified as area-under-curve (AUC) within the range 1.7-3.0 km h-1. Heart rate was recorded, and the number of beats in the same interval was calculated (dHr). Results: O2HbAUC, HHbAUC and dHbAUC differed between diseased and non-diseased legs (P < 0.0001) and exhibited different patterns related to PAD severity according to the ABI value. A compensatory heart rate increase was observed in PAD patients. Compared with the ECD positivity for occlusions/stenoses or multiple plaques, only the receiver-operating characteristic (ROC) analysis of dHbAUC (area = 0.932, P < 0.0001) showed a sensitivity/specificity of 87.6/93.4 for values ≤-197 (LR + LR-: 13.36/0.13). Conclusion: The dynamic NIRS-based test, quantifying muscle metabolic response according to presence and degree of PAD, allows the evaluation of patients with walking disabilities. © 2009 European Society for Vascular Surgery.