Selenium is an essential micronutrient widely present in our diet. It plays its role through the selenoproteins. Previous reports have shown marked variation between individuals in the excretion of this trace element, but the intra-individual variability in selenium excretion has not been specifically investigated. The present study investigates the intra-individual variation in the urinary excretion of selenium in a group of healthy volunteers. We also discuss inter-individual variability trends. Urine samples were collected from healthy volunteers without selenium supplementation twice a day for 7 days and then once a week for an additional 7 weeks. A total of 168 urine samples were collected and analyzed for total selenium and individual selenium species using elemental mass spectrometry and HPLC/mass spectrometry, respectively. We found only modest day-to-day and week-to-week intra-individual variation of selenium excretion. Two commonly reported urine metabolites, selenosugar 1 and selenosugar 3, were detected in all urine samples, and our data suggest that selenosugar 3 is a deacetylated product of selenosugar 1 produced in a manner dependent on selenium intake. Trimethylselenonium displayed no intra-individual variability but considerable inter-individual variability in agreement with the involvement of genetic polymorphisms, as recently reported. Se-methylselenoneine was consistently detected in the urine of all volunteers and was a significant metabolite in one volunteer contributing up to 24% of total urinary selenium. Our data indicate that selenium urinary excretion is consistent within an individual, and that intra-individual variation in selenium excretion is unlikely to complicate future inter-individual variation studies.