Denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and plating on selective agars were used to study variation in the fecal microbiota of rats over time as well as variation between individuals. Investigated rats were either conventional and specific pathogen free (SPF), or human flora associated (HFA). A higher variation (p<0.05) in fecal microbiota over time was observed for HFA than for SPF animals. Analysis of DGGE and T-RFLP profiles of fecal microbiota from SPF and HFA rats revealed that variation over time was less significant than variation between individuals, and that phylogenetic profiles clustered according to gender. These observations should be taken into account when designing future research addressing changes in fecal microbiota.