ObjectiveDiabetic retinopathy in type 2 diabetes is preceded by retinal nerve fiber layer (RNFL) thinning, an index of neurodegeneration. We investigated whether glucose metabolism status (GMS), measures of glycemia, and daily glucose variability (GV) are associated with RNFL thickness over the entire range of glucose tolerance.Research design and methods We used cross-sectional data from The Maastricht Study (up to 5,455 participants, 48.9% men, mean age 59.5 years and 22.7% with type 2 diabetes) to investigate the associations of GMS, measures of glycemia (fasting plasma glucose [FPG], 2-hour post-load glucose [2-h PG], HbA1c, advanced glycation endproducts [AGEs] assessed as skin autofluorescence [SAF]) and indices of daily GV (incremental glucose peak [IGP] and continuous glucose monitoring [CGM]-assessed standard deviation [SD]) with mean RNFL thickness. We used linear regression analyses and, for GMS, P for trend analyses. We adjusted associations for demographic, cardiovascular risk and lifestyle factors, and, only for measures of GV, for indices of mean glycemia. Results After full adjustment, type 2 diabetes and prediabetes (versus normal glucose metabolism) were associated with lower RNFL thickness (standardized beta [95%CI], respectively -0.16[-0.25;-0.08]; -0.05[-0.13;0.03]; Ptrend=0.001). Greater FPG, 2-h PG, HbA1c, SAF, IGP and CGM-assessed SD were also associated with lower RNFL thickness (per SD, respectively -0.05 [-0.08; -0.01]; -0.06 [-0.09; -0.02]; -0.05 [-0.08; -0.02]; -0.04 [-0.07; -0.01]; -0.06 [-0.12; -0.01]; and -0.07 [-0.21; 0.07]).Conclusion In this population-based study, a more adverse GMS and, over the entire range of glucose tolerance, greater glycemia and daily GV were associated with lower RNFL thickness. Hence, early identification of individuals with hyperglycemia, early glucose-lowering treatment, and early monitoring of daily GV may contribute to the prevention of RNFL thinning, an index of neurodegeneration and precursor of diabetic retinopathy.