Objectives Feasibility, validity, and diagnostic accuracy of a non-invasive dynamic ambulatory test were assessed with near infrared spectroscopy (NIRS) evaluating foot perfusion in peripheral arterial disease (PAD). Methods This was a prospective observational study. Eighty PAD patients (63 males, 71 ± 9 years), including 41 patients with coexisting diabetes, participated. Thirteen healthy subjects (8 males, 26 ± 8 years) were also studied by echo colour Doppler providing 160 diseased and 26 non-diseased limbs. Under identical clinostatic conditions, participants performed a 10−repetition toe flexion tests with NIRS probes on the dorsum of each foot; the area under the curve of the oxygenated haemoglobin trace (“toflex area”) was calculated and the ankle–brachial index (ABI) was measured. Time of execution, rate of wrong tests, and adverse reactions were recorded. Within session reliability was assessed by administering the test twice, with a 5 minute interval between tests. The validity was assessed determining whether the toflex area was (a) dependent on the oxygen delivery from the lower limb arteries simulating PAD conditions by a progressive blood flow restriction (40–120% of systolic pressure) in healthy subjects; (b) consistent with the degree of PAD ranked by ABI and correlated with ABI and ankle pressure values in PAD patients. The diagnostic accuracy in detecting PAD was compared with examination using echo colour Doppler ultrasound. Results All tests were rapidly, satisfactorily (<1% mistakes), and safely performed. Toflex area values, superimposable in the two sessions (intra-class correlation coefficient 0.92), were comparable to PAD values following blood flow restriction, consistent with PAD severity, correlated with dorsal pedis artery pressure (r =.21; p =.007) and ABI (r =.65; p <.001) in PAD, but not in the presence of diabetes. Toflex area was similar to echo colour Doppler for detecting PAD following receiver operating characteristic curve analysis (area = 0.987, p <.001; toflex area values ≤ −28 arbitrary units, sensitivity/specificity 95.6/100). Conclusion The toe flexion test enables ambulatory assessment of foot perfusion and PAD detection, even in the presence of non-measurable ABI or diseases affecting the microcirculation.