Background: The best treatment for end-stage renal disease (ESRD) is kidney transplantation. Twenty-seven percent of transplantations in Norway are from living donors. Recent studies have shown an increased risk of ESRD and increased mortality in donors. The aim of this study was to determine if the levels of the new biomarkers neutrophil gelatinase-associated lipocalin (NGAL), soluble Klotho (sKlotho), and fibroblast growth factor 23 (FGF23) are changed in kidney donors with normal kidney function defined as an estimated glomerular filtration rate (eGFR) >60 ml/min/1.73 m2 compared to patients with chronic kidney disease (CKD) stages 3-5 and healthy controls. Methods: This is a cross-sectional, observational, single-center study including 35 kidney donors with an eGFR ≥60 ml/min/1.73 m2 5 years after donation, 22 patients with CKD stage 3 (eGFR 30-59 ml/min/1.73 m2), 18 patients with CKD stage 4 (eGFR 15-29 ml/min/1.73 m2), 20 patients with CKD stage 5 (eGFR <15 ml/min/1.73 m2), and 35 controls comparing levels of biomarkers in long-term kidney donors with those in CKD patients and healthy controls. Results: The level of log NGAL was significantly higher in donors than in healthy controls (2.02 ± 0.10 vs. 1.89 ± 0.10 ng/ml; p < 0.001), and the level increased with declining kidney function. The log FGF23 level was nonsignificantly higher in donors than in controls, but it significantly increased with declining kidney function. The log sKlotho levels were significantly lower in patients with CKD stages 4 and 5 than in controls, but no difference was revealed between controls and donors. Conclusion: Kidney donors have significantly higher levels of NGAL than healthy controls after a median of 15 years (range 5-38). NGAL could be a valuable diagnostic marker in the future. FGF23 and sKlotho were not significantly different between donors and controls.