Background: The predictive value of acute kidney injury (AKI) urinary biomarkers may depend on the time interval following tubular injury, thereby explaining in part the heterogeneous performance of these markers that has been reported in the literature. We studied the influence of timing on the predictive values of tubular proteins, measured before the rise of serum creatinine (SCr) in critically ill, non-septic patients. Methods: Seven hundred adult critically ill patients were prospectively included for urine measurements at four time-points prior to the rise in serum creatinine (T = 0, -16, -20 and -24 h). Patients with sepsis and or AKI at ICU entry were excluded. The urinary excretion of the proteins, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), which are up-regulated in the distal and proximal tubules, respectively, were measured as well as the constitutive cytopla Results: Five hundred and forty-three subjects were eligible for further analyses; however, 49 developed AKI in the first 48 h. Both NGAL (P = 0.001 at T = -24 vs. non-AKI patients) and KIM-1 (P < 0.0001 at T = 0 vs. non-AKI patients) concentrations gradually increased until AKI diagnosis, whereas pi- and alpha-GST peaked at T = -24 before AKI (P = 0.006 and P = 0.002, respectively vs. non-AKI patients) and showed a rapid decline afterwards. The predictive values at T = -24 prior to AKI were mod Conclusions: NGAL, KIM-1, pi- and alpha-GST displayed unique and mutually incomparable time dependent characteristics during the development of non-sepsis related AKI. Therefore, the time-relationship between the biomarker measurements and the injurious event influences the individual test results.