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The 24-h urine sample is considered as the most reliable material for testing many but not necessarily all constituents in urine. However, its collection is tedious for both patients and research participants. The aim of this study was to compare concentrations of essential elements calcium (Ca), magnesium (Mg), sodium (Na), potassium (K), and zinc (Zn) in 24-h and spot urine samples.Urine samples were collected from 143 generally healthy women, aged 30–79 years. Fasting spot urine was collected immediately after the end of the 24-h collection, therefore being of the same content as the first morning urine which ended the 24-h collection. Elements were analyzed by flame atomic absorption/emission spectrometry and expressed as mg/g and/or mmol/mol of creatinine (Cr). Spearman rank order correlations between 24-h and spot urine were carried out for each element. Ratios of elements in 24-h to spot urine samples were calculated to estimate the element-proportion of spot in the 24-h sample.All coefficients of correlation between 24-h and spot urine of measured elements and Cr were significant (p<0.05): Zn (0.637), Mg (0.623), Ca (0.603), Na (0.452), K (0.396), and Cr (0.217). Ratios of 24-h to spot urine samples for each element (except K) were similar and close to 2, indicating uniform proportion of elements from spot urine sample in the 24-h sample. In addition, a high correlation between various pairs of elements was obtained in both 24-h and spot urine; the highest being between Na/Ca (0.435) and (0.578), respectively. This is in accordance with theoretical presumptions and previous findings regarding those relationships.Although replacing burdensome 24-h urine collection with spot urine sampling might not provide the solution in all cases, our results show that for the elements analyzed, spot urine could be a reliable alternative.