Prepulse inhibition (PPI) and habituation of the acoustic startle response (ASR) are widely used biological markers in the study of psychiatric disorders and have been shown to be homologous across species. Previous studies in humans suggested that PPI is a stable and reliable measure between test sessions, but that PPI decreases within sessions. The purpose of this study was to explore the short- and long-term decrease in PPI as a potential confound in the measurement and interpretation of PPI. We investigated the progression of PPI and habituation of ASR in three test sessions spaced 4 weeks apart in a group of 20 healthy participants. Analysis revealed a significant decrease in the percent PPI within and between the test sessions. Nevertheless, PPI was reliable across three test sessions, indicating that the significant attenuation of PPI over time was a consistent phenomenon. These results suggest that PPI exhibits short- and long-term attenuation.