BACKGROUND Flexible endoscopes are currently reused following cleaning and high-level disinfection. Contamination has been found on endoscopes, and infections have been linked to gastrointestinal, respiratory, and urologic endoscopes. METHODS This longitudinal study involved visual inspections with a borescope, microbial cultures, and biochemical tests for protein and adenosine triphosphate to identify endoscopes in need of further cleaning or maintenance. Three assessments were conducted over a 7-month period. Control group endoscopes reprocessed using customary practices were compared with intervention group endoscopes subjected to more rigorous reprocessing. RESULTS At final assessment, all endoscopes (N = 20) had visible irregularities. Researchers observed fluid (95%), discoloration, and debris in channels. Of 12 (60%) endoscopes with microbial growth, 4 had no growth until after 48 hours. There were no significant differences in culture results by study group, assessment period, or endoscope type. Similar proportions of control and intervention endoscopes (~20%) exceeded postcleaning biochemical test benchmarks. Adenosine triphosphate levels were higher for gastroscopes than colonoscopes (P = .014). Eighty-five percent of endoscopes required repair due to findings. CONCLUSIONS More rigorous reprocessing was not consistently effective. Seven-day incubation allowed identification of slow-growing microbes. These findings bolster the need for routine visual inspection and cleaning verification tests recommended in new reprocessing guidelines.