The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at -80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or -80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or -80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.