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The water deprivation test is the gold standard test to differentiate central or nephrogenic diabetes insipidus (DI) from primary polydipsia (PP) in patients with polyuria and polydipsia. Few studies have addressed the diagnostic performance of this test. The aim of this retrospective cohort study was to evaluate the diagnostic performance of the standard water deprivation test, including plasma arginine vasopressin (AVP) measurements, in 40 consecutive patients with polyuria. We compared initial test results with the final clinical diagnosis, i.e., no DI, central DI, or nephrogenic DI. The median length of follow-up was 8 years. In a subset of ten patients, the novel marker copeptin (CP) was measured in plasma. Using the final diagnosis as a gold standard, a threshold for urine osmolality of >800 mOsmol/kg after water deprivation yielded a sensitivity and specificity of 96 and 100%, respectively, for diagnosing PP. Sensitivity increased to 100% if the cut-off value for urine osmolality was set at 680 mOsmol/kg. Plasma AVP levels did not differ between patient groups and did not differentiate among central DI, nephrogenic DI, or PP. In all three patients with central DI, plasma CP was <2.5 pmol/l with plasma osmolality >290 mOsmol/kg, and >2.5 pmol/l in patients without DI. The optimal cut-off value for differentiating PP from DI during a water deprivation test was urine osmolality >680 mOsmol/kg. Differentiating between central and nephrogenic DI should be based on clinical judgment as AVP levels did not discriminate.