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INTRODUCTION: Hyperspectral imaging (HSI) is a noninvasive technique for transcutaneous measurements of tissue perfusion. This study (1) provides a review of the current literature on HSI for tissue perfusion measurements of the lower leg and (2) introduces a standardized measurement protocol for HSI measurements with a portable system. EVIDENCE ACQUISITION: A literature search was performed for studies on tissue perfusion measurements with HSI in the lower extremity. A standardized protocol was developed to perform HSI measurements in 43 healthy volunteers at the plantar side of the foot and at the lateral side of the calf, with 3 consecutive hyperspectral images at each location. EVIDENCE SYNTHESIS: The literature review identified 9 studies, including 2 of healthy volunteers. 4 of patients with diabetes mellitus, and 3 of patients with peripheral arterial disease. In 5 of 7 patient studies, HSI values were associated with severity of disease or wound healing. In our study, the healthy volunteers' I ISI values for oxyhemoglobin, deoxyhemoglobin, and oxygen saturation were (mean +/- SD) 82.8 +/- 24, 55.7 +/- 15.7, and 59.2 +/- 11.7, respectively, at the plantar surface of the foot, and 40.8 +/- 11, 38.0 +/- 7.8, and 51.7 +/- 10.5, respectively, at the lateral side of the calf. HSI values differed significantly between the calf and plantar locations. Intraoperator reliability between the 3 consecutive images ranged from 81% to 89%. CONCLUSIONS: Limited evidence indicates that HSI is associated with severity of peripheral arterial disease and diabetes mellitus, and with wound healing. Hyperspectral images with a portable system can be taken with high precision when a standardized measurement protocol is used. However, differences exist at several locations at the lower extremity, so each measurement location should be used as its own reference when consecutive measurements are performed during follow-up. More studies with larger patient cohorts should be performed before HSI can be incorporated as standard tool in the diagnostic armamentarium of the vascular specialist.