Urine and cerebrospinal fluid (CSF) are two important biofluids used for disease biomarker discovery. For differential proteomic analysis, it is essential to evaluate individual and gender variations. In this study, we characterized urinary and CSF proteomes of 14 healthy volunteers with regard to individual and gender variations using 2DLC-MS/MS analysis and 8-plex iTRAQ quantification. A total of 968/512 urinary/CSF proteins were identified, with 406/280 quantified in all individuals. The median inter-individual coefficients of variation (CVs) were 0.262 and 0.183 for urinary and CSF proteomes, respectively. Cluster analysis showed that male and female urinary proteomes exhibited different patterns, though CSF proteome showed no remarkable gender differences. In comparison with CSF proteome, urinary proteome showed higher individual variation. Further analysis revealed that individual variation was not correlated with protein abundance. The minimum sample size for proteomic analysis with a 2-fold change was 10 (4/5 for males/females using iTRAQ quantification) for urinary or 8 for CSF proteome. Intracellular proteins leaked from exfoliative cells tended to have higher CVs, and extracellular proteins secreted from urinary tract or originating from plasma tended to have lower CVs. The above results might be beneficial for differential proteomic analysis and biomarker discovery.