Significant lower limb arterial obstruction is usually detected by Doppler ankle-brachial pressure index (ABPI) measurement. However, ABPI is non-contributory in cases of diabetic medial sclerosis or calcifications and is unsuitable for the detection of small vessel involvement. Thallium-201, a perfusion agent, is frequently used for the investigation of coronary artery disease, and whole-body (201)Tl scintigraphy (WBS) has also been reported to be useful in the assessment of peripheral artery disease (PAD). Thus, we evaluated the clinical feasibility of simultaneous myocardial and lower limb perfusion assessment. WBS was performed after treadmill exercise and myocardial scintigraphy, and again 4 h later. Calf (201)Tl fractional activities (percentage of whole-body (201)Tl uptake) were calculated. We determined a threshold value of normal post-exercise calf (201)Tl uptake (mean of the (201)Tl fractional uptakes minus 2 SD) in a control group of nine healthy volunteers. We checked its accuracy in a pilot group of 25 diabetic patients with proven PAD. This method permitted the detection of lower limb perfusion abnormalities in 38% of 47 asymptomatic diabetic patients with no evidence of PAD. In conclusion, for asymptomatic diabetic patients, whole-body (201)Tl scintigraphy after a treadmill test seems an efficient method of showing lower limb perfusion abnormalities not detected by ABPI measurement. It allows the evaluation of vascular status with no additional inconvenience for patients when performed after myocardial scintigraphy.