The rotational variance dependence of diffusion tensor imaging (DTI) derived parameters on the number of diffusion weighting directions (N) has been investigated by several Monte Carlo simulation studies. However, the dependence of fractional anisotropy (FA) and mean diffusivity (MD) maps on N, in terms of accuracy and contrast between different anatomical structures, has not been assessed in detail. This experimental study further investigated in vivo the effect of the number of diffusion weighting directions on DTI maps of FA and MD. Human brain FA and MD maps of six healthy subjects were acquired at 1.5T with varying N (6, 11, 19, 27, 55). Then, FA and MD mean values in high (FAH, MDH) and low (FAL, MDL) anisotropy segmented brain regions were measured. Moreover, the contrast-to-signal variance ratio (CVRFA, CVRMD) between the main white matter and the surrounding regions was calculated. Analysis of variance showed that FAL, FAH and CVRFA significantly (p < 0.05) depend on N. In particular, FAL decreased (6%-11%) with N, whereas FAH (1.6%-2.5%) and CVRFA (4%-6.5%) increased with N. MDL, MDH and CVRMD did not significantly (p>0.05) depend on N. Unlike MD values, FA values significantly vary with N. It is noteworthy that the observed variation is opposite in low and high anisotropic regions. In clinical studies, the effect of N may represent a confounding variable for anisotropy measurements and the employment of DTI acquisition schemes with high N (> 20) allows an increased CVR and a better visualization of white matter structures in FA maps.