The gas exchange surfaces of the lungs is an attractive target for local drug delivery because this area represents the pathological site for a number of lung diseases, and for systemic drug delivery as it is a noninvasive route avoiding the enzyme rich GI tract. Nucleic acid-based drugs, e.g. RNA interference (RNAi) herapeutics like small interfering RNA (siRNA), are promising next-generation biopharmaceuticals due to their unprecedented specificity and potency. For non-invasive administration routes such as the intrapulmonary route, drugs in this class may be loaded into delivery systems to enhance intracellular delivery and to protect the cargo against enzymatic degradation. The deposition target for these formulations may be the deep lung, but deposition in the conducting airways must also be considered during development of inhalable dosage forms. Potential consequences of deposition in the upper airways is that the nanoformulations are cleared before absorption and/or may interfere with the rheological properties of mucus, eventually resulting in altered physiological mucociliary clearance parameters. The purpose of this study was to investigate whether different nanoformulation had different propensities to alter mucus rheology. We compared the effect of three different nanoformulations loaded with the same siRNA cargo on the rheological properties of a model mucus matrix. We found that the presence of nanoformulations or naked siRNA increased the elastic modulus of the mucus matrix, but the phase angle, which determines the functional properties of mucus, remained essentially unchanged. There were no statistically significant difference between the nanocarriers tested in this study. This suggests that potential mucus rheological consequences of off target deposition need not be a major consideration when selecting siRNA carrying nanoformulations.