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The main cause of endoscopy-associated infections is failure to adhere to reprocessing guidelines. More information about factors impacting compliance is needed to support the development of effective interventions. The purpose of this multisite, observational study was to evaluate reprocessing practices, employee perceptions, and occupational health issues. Data were collected utilizing interviews, surveys, and direct observation. Written reprocessing policies and procedures were in place at all five sites, and employees affirmed the importance of most recommended steps. Nevertheless, observers documented guideline adherence, with only 1.4% of endoscopes reprocessed using manual cleaning methods with automated high-level disinfection versus 75.4% of those reprocessed using an automated endoscope cleaner and reprocessor. The majority reported health problems (i.e., pain, decreased flexibility, numbness, or tingling). Physical discomfort was associated with time spent reprocessing (p = .041). Discomfort diminished after installation of automated endoscope cleaners and reprocessors (p = .001). Enhanced training and accountability, combined with increased automation, may ensure guideline adherence and patient safety while improving employee satisfaction and health.