Abstract The rheology of sputum is viewed as a powerful emerging biophysical marker for monitoring muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). However, there is no unified practice to process sputa from collection to analysis, which can lead to highly variable, and sometimes inconsistent results. The main objective of this study is to bring light into the handling of sputum samples to establish a standardised and robust protocol before rheological measurements. Sputum collected from 22 CF and 10 NCFB adults, was divided into control (vortexed and fresh: non-heated and non-frozen) and three treated conditions (either non-vortexed, heated or frozen). In addition, 6 CF expectorations were used to study the dynamics of ageing over 24 h. Sputum’s mechanical properties were measured with a rotational rheometer to obtain their properties at rest, elastic (

$$G'$$ G ′ ) and viscous moduli ( $$G''$$ G ′ ′ ), and at the onset of flow, critical deformation ( $$\gamma _c$$ γ c ) and critical stress ( $$\sigma _c$$ σ c ). We demonstrate that heating sputum is completely destructive while freezing sputa at $$-80\,\,^{\circ }\hbox {C}$$ - 80 ∘ C has no discernible effect on their rheology. We also show that the variability of rheological measurements largely resulted from the sample’s macroscopic heterogeneity, and can be greatly reduced by non-destructive vortex homogenisation. Finally, we observed contrasted ageing effects as a fonction of purulence: while the viscoelasticity of purulent samples reduced by half within 6 h after collection, semi-purulent samples did not evolve. These results guide towards a robust unified protocol for simple sputum handling in rheometry. We therefore suggest to vortex and snap freeze sputum samples immediately after collection when direct testing is not possible.