To investigate if variations in concentrations occur at different levels within urine tanks and to evaluate possible consequences thereof, urine samples were collected from four collection tanks and one storage tank of different urine separating sewerage systems. Plant nutrients and metals were found to concentrate in the sediment at the bottom of the tanks. Also, densities of indicator bacteria were higher in the bottom layer, probably due to adsorption to sedimented particles. The differences in concentration at different levels became more apparent during storage of the urine mixture. After four months storage concentrations of the investigated indicator bacteria, except clostridia, were below detection limits at all levels. Considering the variability in nutrient concentration, samples from the middle level correspond well to the average composition in the tanks and can be used to calculate urine application rates. When estimating hygienic risks with the reuse of human urine the concentration variability also needs to be considered.