There are indications that the excretion of sodium (Na+) and potassium (K+) into urine is affected by the body’s circadian rhythm. Mill et al. (2012) reported that the Na+/K+ ratio was significantly higher in urine samples taken at night compared with day, but the mechanism underlying this remained unknown. Aldosterone regulates the renal reabsorption of Na+ and the secretion of K+ (Wiederholt et al., 1972). We hypothesize that aldosterone may play a role in the diurnal and nocturnal excretions of Na+ and K+. We investigated the Na+/K+ ratios in 24-hour (24h) urine and in different-timed urine samples; and assessed the concentrations of aldosterone in serum, and 24h and 12h (7am-7pm, D; and 7pm-7am, N) urine samples. Ethical approval was obtained for a total of 25 healthy subjects, 11 females and 14 males aged 20-30 years, who were included in the study. Subjects were on their regular diet and were instructed to avoid alcohol three days before and during the period of sample collection. Blood samples, 12h-D and 12h-N urine samples were collected. 24h urine samples were obtained from the two 12h samples. Urine and plasma concentrations of Na+ and K+ were analyzed using flame photometry; and the urine and serum aldosterone concentrations were analyzed using the enzyme immunoassay method (DRG International, Inc., USA). Urinary Na+/K+ratios were calculated by dividing the molar concentrations of Na+ by that of K+ in urine, both expressed in mmol/L. Results are presented as means ± S.E.M., and analyzed using unpaired Students t-test. Significantly higher Na+/K+ ratios were observed in the 12h-N urine compared with the 12h-D urine in the male subjects (4.18±0.22 vs 2.92±0.24, p<0.05). Aldosterone concentrations in the serum (ng/dl); and in the 24h urine (ug/24h) and the 12h-D and12h-N urine (ug/12h) samples were similar in both male and female subjects. Correlation analysis was used to examine the relationship between different groups. No significant correlation was observed between urinary aldosterone concentrations and the 12h urinary Na+/K+ ratios. However, significant correlation coefficients were found between Na+/K+ ratios in 24h and 12h urine samples (male, 24h and 12h-D: r2=0.4289, 24h and 12h-N: r2=0.2750, n= 14, p<0.05; and female, 24h and 12h-D: r2=0.5341 and 24h and 12h-N: r2=0.4313, n=11, p<0.05). We conclude that urinary Na+/K+ ratio is affected by the time of urine collection. Also, urine samples collected within the 12h-D or 12h-N period may be used to estimate the 24h urinary Na+/K+ ratio. Furthermore, our results suggest that aldosterone may not be responsible for the diurnal and nocturnal variations in the excretion of Na+ and K+ into urine.