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BACKGROUND & AIMS Oral lutein (L) and zeaxanthin (Z) supplementation enhances macular pigment optical density (MPOD) and plays a protective role in the development of age-related macular degeneration (AMD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a novel in vivo retinal imaging method that has been shown to correlate to classical MPOD measurements and might contribute to a metabolic mapping of the retina in the future. Our aim was to show that oral supplementation of L and Z affects the FLIO signal in a positive way in patients with AMD. METHODS This was a prospective, single center, open label cohort study. Patients with early and intermediate AMD received oral L and Z supplementation during three months, and were observed for another three months after therapy termination. All visits included measurements of clinical parameters, serum L and Z concentration, MPOD measurements using heterochromatic flicker photometry, dual wavelength autofluorescence imaging, and FLIO. Correlation analysis between FLIO and MPOD were performed. RESULTS Twenty-one patients completed the follow up period. Serum L and Z concentrations significantly increased during supplementation (mean difference 244.8 ng/ml; 95% CI: 81.26-419.9, and 77.1 ng/ml; 95% CI: 5.3-52.0, respectively). Mean MPOD units significantly increased (mean difference 0.06; 95% CI: 0.02-0.09; at 0.5°, 202; 95% CI: 58-345; at 2°, 1033; 95% CI: 288-1668; at 9° of eccentricity, respectively) after three months of supplementation with macular xanthophylls, which included L and Z. Median FLIO lifetimes in the foveal center significantly decreased from 277.3 ps (interquartile range 230.2-339.1) to 261.0 ps (interquartile range 231.4-334.4, p = 0.027). All parameters returned to near-normal values after termination of the nutritional supplementation. A significant negative correlation was found between FLIO and MPOD (r2 = 0.57, p