BackgroundLevels of urinary biomarkers, such as kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), can determine the risk of renal damage and predict the outcome of multiple kidney diseases, including transplant rejection. Studies of urinary biomarkers frequently use urine that has been stored frozen before analysis without taking into account the potential of biomarker degradation during storage. Study DesignObservational study. Setting & Participants39 kidney transplant patients visiting an outpatient clinic. PredictorUrine storage conditions (4°C and −80°C with or without protease inhibitors) and centrifugation after thawing of samples. OutcomesUrinary concentrations of NGAL and KIM-1. MeasurementsNGAL and KIM-1 were measured with commercially available enzyme-linked immunosorbent assay kits. ResultsUrinary NGAL and KIM-1 concentrations were stable in urine up to 48 hours when stored at 4°C and up to 6 months when stored at −80°C, independent of the addition of protease inhibitors. A centrifugation step prior to biomarker measurement did not change urinary biomarker concentrations of urine stored at −80°C. LimitationsOnly KIM-1 and NGAL were measured, which possibly limits the relevance of the findings when making determinations about other urinary biomarkers. ConclusionsThis study recommends that urine samples collected for the assessment of KIM-1 or NGAL are immediately cooled to 4°C and subsequently frozen at −80°C within 2 days. At −80°C, both KIM-1 and NGAL concentrations are stable for at least 6 months.