Background/Objectives While daily hydration is best assessed in 24-h urine sample, spot sample is often used by health care professionals and researchers due to its practicality. However, urine output is subject to circadian variation, with urine being more concentrated in the morning. It has been demonstrated that afternoon spot urine samples are most likely to provide equivalent urine concentration to 24-h urine samples in adults. The aim of the present study was to examine whether urine osmolality (UOsm) assessed from a spot urine sample in specific time-windows was equivalent to 24-h UOsm in free-living healthy children. Subjects/Methods Among 541 healthy children (age: 3-13 years, female: 45%, 77% non-Hispanic white, BMI:17.7 ± 4.0 kg m −2), UOsm at specific time-windows [morning (0600-1159), early afternoon (1200-1559), late afternoon (1600-1959), evening (2000-2359), overnight (2400-0559), and first morning] was compared with UOsm from the corresponding pooled 24-h urine sample using an equivalence test. Results Late afternoon (1600-1959) spot urine sample UOsm value was equivalent to the 24-h UOsm value in children (P < 0.05; mean difference: 62 mmol kg −1 ; 95% CI: 45-78 mmol kg −1). The overall diagnostic ability of urine osmolality assessed at late afternoon (1600-1959) to diagnose elevated urine osmolality on the 24-h sample was good for both cutoffs of 800 mmol kg −1 [area under the curve (AUC): 87.4%; sensitivity: 72.6%; specificity: 90.5%; threshold: 814 mmol kg −1 ] and 500 mmol kg −1 (AUC: 83.5%; sensitivity: 75.0%; specificity: 80.0%; threshold: 633 mmol kg −1). Conclusion These data suggest that in free-living healthy children, 24-h urine concentration may be approximated from a late afternoon spot urine sample. This data will have practical implication for health care professionals and researchers.

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