The respiratory epithelium is a polarised layer at the interface between the outside environment and deeper lung structures, overlaid by the epithelial lining fluid (ELF). This provides a mechanical and immunological barrier to inhaled particulates, such as viruses. Human respiratory syncytial virus (hRSV) is a major cause of disease in humans, and targets the respiratory epithelium. However, little is known of the disruption of the ELF proteome in the context of virus-driven respiratory illnesses. To address this, a proteomics approach was combined with an ex-vivo human airway epithelial model (HAE) to investigate the apical and basolateral secretome in hRSV-infected cultures. This demonstrated that several apically- and basolaterally-restricted proteins were subsequently secreted in both directions upon infection, while a number of proteins saw their apical/basolateral abundance ratios significantly altered. Furthermore, another 35 proteins were uniquely identified after hRSV treatment. Importantly, some of these changes were correlated in nasal aspirates (NA) from children with and without hRSV. This study showed that hRSV could affect airway secretions, and disrupted the directionality of the respiratory epithelium.