AbstractVentilator-associated pneumonia (VAP) is a common hospital-acquired infection, leading to high morbidity and mortality. Currently, bronchoalveolar lavage (BAL) is utilized in hospitals for VAP diagnosis and guiding treatment options. While BAL collection procedures are invasive, alternatives such as endotracheal aspirates (ETA) may be of diagnostic value, however, their utility has not been thoroughly explored. Longitudinal ETA and BAL were collected from 16 intubated patients up to 15 days, of which 11 developed VAP. We conducted a comprehensive LC-MS/MS based proteome and metabolome characterization of longitudinal ETA and BAL to detect host and pathogen responses to VAP infection. We discovered a diverse ETA proteome of the upper airways reflective of a rich and dynamic host-microbe interface. Prior to VAP diagnosis by microbial cultures from BAL, patient ETA presented characteristic signatures of reactive oxygen species and neutrophil degranulation, indicative of neutrophil mediated pathogen processing as a key host response to the VAP infection. Along with an increase in amino acids, this is suggestive of extracellular membrane degradation resulting from proteolytic activity of neutrophil proteases. Days prior to VAP diagnosis, detection of pathogen peptides with species level specificity in ETA may increase specificity over culture-based diagnosis. Our findings suggest that ETA may provide early mechanistic insights into host-pathogen interactions associated with VAP infection and therefore facilitate its diagnosis and treatment.Graphical abstract