BACKGROUND Multiple outbreaks of carbapenem-resistant enterobacteriaceae (CRE) and other multi-drug resistant organisms (MDROs), have been associated with flexible endoscopes resulting in unacceptable patient mortality and morbidity. Evidence highlights the importance of effective cleaning to achieve effective high-level disinfection (HLD). The objective of this study is to quantify clinical soil removal from endoscopes through manual cleaning. METHODS This simulated-use study (85 individual trials) measured adenosine-triphosphate (ATP) and protein levels found in the suction/biopsy channel of OlympusTJF-160F duodenoscopes inoculated with an artificial test soil, after partial and complete manual cleaning. Three partial manual cleaning modes of critical importance were identified for the study: 1) incomplete brushing of the suction/biopsy channel, 2) omitting aspiration of the suction/biopsy channel, 3) cleaning an endoscope where soil dried for 24 hours. Analysis used one-way ANOVAs to determine statistical differences. p-values RESULTS Elevated ATP levels above the cleanliness criterion value of 200 relative light units (RLUs) (M.J. Alfa, et al.; AJIC; 41; (2013); 245-248 and 249-253; ANSI/AAMI ST91:2015) were detected for all the partial cleaning modes studied when compared to complete cleaning (p-value CONCLUSIONS This study highlights the importance of complete manual cleaning and finds that incomplete cleaning of duodenoscopes results in 6 to 14 times larger amounts of residual soil. Protein is not detected in partially cleaned duodenoscopes when ATP clearly indicates elevated amounts of remaining soil. The methodology and results of this study can be used to establish a better cleanliness criterion for monitoring the effectiveness of manual cleaning.