Fecal sample collection is an important influential factor for DNA-based gut microbiota study. It is controversial whether the microbiome detected in fecal sample collected at one random day could fully represent the gut microbial community. The aim of the study is to figure out whether the use of fecal sample mixture collected at consecutive 5 days could more accurately represent gut microbial community. 1- and 5-day fecal samples were collected from 8 healthy adults and analyzed by 16S rRNA sequence. Our results indicated that both 1-day fecal samples and 5-day samples exhibited relatively high repeatability. The relative abundance of majority of bacterial taxa did not changed between 1-day fecal samples and 5-day fecal samples. However, the alpha diversity of 5-day fecal samples was higher than that of 1-day fecal samples. When the aims of studies are to analyze the relative abundance of specific OTUs among subjects, fecal samples collected at one day could be used. When microbial diversity is one of essential factors to be analyzed, the use of 5-day fecal samples may be more recommended.