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1H NMR-based metabonomics has shown considerable potential as a means of rapidly detecting and characterizing a myriad of metabolic components. The 1H NMR spectra obtained for human urine samples were visually inspected and analyzed using principal component analysis (PCA). Urine samples were collected eight times daily (8:00, 10:00, 12:00, 14:00,18:00, 20:00) from healthy volunteers with a standard diet and lifestyle for 2 consecutive days. Separation of urinary data according to time of sampling collection was achieved. The fluctuations of creatinine, citrate and observed between urine samples collected during the different time of day may be related to hormone levels. In addition, gender-difference was also investigated and the PCA score plots revealed several metabolites that were considered significant for distinguishing between male and female: creatinine, glycine and citrate. These results showed the value of metabonomic methods in the investigation of physiological variation and rhythms, and provided suggestions to collect significative metabonomics data in clinical studies.