When selecting the least biased exposure surrogate, for example, the concentration of a biomarker in a urine sample, information on variability must be taken into consideration. We used mixed-effects models to estimate the variability and determinants of urinary cadmium (U-Cd) excretion using spot urine samples collected at six fixed times during 2 days about 1 week apart, from 24 healthy non-smokers. The urine samples were analysed for U-Cd, the concentrations were adjusted for dilution, and the excretion rates were calculated. Between-individual variability dominated the total variability for most measures of U-Cd excretion, especially for 24 h urine and first morning samples. The U-Cd excretion showed a circadian rhythm during the day, and time point of sampling was a significant factor in the mixed-effects models, thus a standardised sampling time, such as first morning urine samples, needs to be applied. Gender, urinary flow rate, age, and urinary protein excretions were also significant determinants for U-Cd excretion. The choice of biomarker for U-Cd excretion was found to be more important in individually-based studies of exposure-response relationships than in studies of comparing Cd levels of groups. When planning a study, this variability of U-Cd in spot samples must be acknowledged.