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High fluid intake has been universally recommended for kidney stone prophylaxis. We evaluated 24-h urine osmolality regarded as the best biomarker of optimal hydration and upper metastable limit osmolality after water evaporation from urine sample to the onset of spontaneous crystallization and its usefulness as a new risk index that would describe an individual lithogenic potential. We collected 24-h urine from 257 pediatric patients with kidney stones and 270 controls. After volume and osmolality assessment, the urine samples were subjected to volume reduction in vacuum rotavapor continued to the onset of an induced urinary crystallization. The upper metastable limit osmolality of urine sample was calculated based on its initial osmolality value and the amount of water reduction. Pediatric stone formers presented with higher urine volume and lower urine osmolality than healthy controls. Despite that, their urine samples required much lower volume reduction to induce the spontaneous crystallization than those of controls. The ROC analysis revealed an AUC for the upper metastable limit osmolality of 0.9300 (95% CI 0.9104-0.9496) for distinguishing between stone formers and healthy subjects. At the cutoff of 2696 mOsm/kg, the test provided sensitivity and specificity of 0.8638 and 0.8189, respectively. 24-h urine osmolality provided the information about current hydration status, whereas evaporation test estimated the urinary potential to crystalize dependent on urine composition. Upper metastable limit osmolality may estimate the individual lithogenic capability and identify people at risk to stone formation when exposed to dehydration. [ABSTRACT FROM AUTHOR]