Although the median urinary iodine concentration (UIC) is a good indicator of iodine status in populations, there is no established biomarker for individual iodine status. If the UIC were to be used to assess individuals, it is unclear how many repeat urine collections would be needed and if the collections should be spot samples or 24-h samples. In a prospective, longitudinal, 15-mo study, healthy Swiss women (n = 22) aged 52-77 y collected repeated 24-h urine samples (total n = 341) and corresponding fasting, second-void, morning spot urine samples (n = 177). From the UIC in spot samples, 24-h urinary iodine excretion (UIE) was extrapolated based on the age- and sex-adjusted iodine:creatinine ratio. Measured UIE in 24-h samples, estimated 24-h UIE, and UIC in spot samples were (geometric mean ± SD) 103 ± 28 μg/24 h, 86 ± 33 μg/24 h, and 68 ± 28 μg/L, respectively, with no seasonal differences. Intra-individual variation (mean CV) was comparable for measured UIE (32%) and estimated UIE (33%). The CV tended to be higher for the spot UIC (38%) than for the estimated 24-h UIE (33%) (P = 0.12). In this population, 10 spot urine samples or 24-h urine samples were needed to assess individual iodine status with 20% precision. Spot samples would likely be preferable because of their ease of collection. However, the large number of repeated urine samples needed to estimate individual iodine status is a major limitation and emphasizes the need for further investigation of more practical biomarkers of individual iodine status.