OBJECTIVE Urinary cadmium (U-Cd) sampling can be done either by 24-h urine or spot urine sampling, and adjustment for dilution is usually needed. The choice of sampling period and adjustment technique could, however, potentially induce bias. The aim of the study was to compare 24-h urine and spot urine sampling and two dilution adjustment techniques, when assessing U-Cd. METHODS Separate 24-h urine (U24) and timed overnight spot urine (UON) samples were collected from 152 healthy kidney donors. U-Cd, creatinine concentration (U-Crea) and specific gravity (SG) were analysed. Differences between U24 and UON samples were tested using paired t test, and the effect of urinary flow rate (UF) was assessed by linear regression. RESULTS The cadmium excretion rate (U-Cd/h) was lower in the UON than in U24 samples (mean 0.017 μg/h vs. 0.021 μg/h; p < 0.001). This decrease was found also for the creatinine-adjusted U-Cd (U-CdCrea) (mean 0.36 μg/gC and 0.41 μg/gC; p < 0.001). For U-Cd adjusted for specific gravity (U-CdSG), the difference was reversed, but not statistically significant. The creatinine excretion rate (U-Crea/h) decreased at low UF, especially in the UON. CONCLUSIONS Since U-Cd/h was lower in UON than in U24 samples, the former will underestimate the true Cd excretion. This was seen for U-CdCrea but not for U-CdSG. However, it may be an advantage that the U-CdSG is similar, irrespective of sampling strategy. At low UF, U-CdCrea will be biased upwards. Whether U24 or UON samples adjusted for U-Crea or SG best reflect kidney-Cd is still unknown.