Purpose:Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) is gaining popularity for the quantification of tissue perfusion, including foot perfusion in patients with lower extremity arterial disease (LEAD). However, the absolute fluorescence intensity is influenced by patient—and system-related factors limiting reliable and valid quantification. To enhance the quality of quantitative perfusion assessment using ICG NIR fluorescence imaging, normalization of the measured time-intensity curves seems useful.Materials and Methods:In this cohort study, the effect of normalization on 2 aspects of ICG NIR fluorescence imaging in assessment of foot perfusion was measured: the repeatability and the region selection. Following intravenous administration of ICG, the NIR fluorescence intensity in both feet was recorded for 10 mins using the Quest Spectrum platform®. The effect of normalization on repeatability was measured in the nontreated foot in patients undergoing unilateral revascularization preprocedural and postprocedural (repeatability group). The effect of normalization on region selection was performed in patients without LEAD (region selection group). Absolute and normalized time-intensity curves were compared.Results:Successful ICG NIR fluorescence imaging was performed in 54 patients (repeatability group, n = 38; region selection group, n = 16). For the repeatability group, normalization of the time-intensity curves displayed a comparable inflow pattern for repeated measurements. For the region selection group, the maximum fluorescence intensity (Imax) demonstrated significant differences between the 3 measured regions of the foot (P= .002). Following normalization, the time-intensity curves in both feet were comparable for all 3 regions.Conclusion:This study shows the effect of normalization of time-intensity curves on both the repeatability and region selection in ICG NIR fluorescence imaging. The significant difference between absolute parameters in various regions of the foot demonstrates the limitation of absolute intensity in interpreting tissue perfusion. Therefore, normalization and standardization of camera settings are essential steps toward reliable and valid quantification of tissue perfusion using ICG NIR fluorescence imaging.