To test the impact of cholesterol on membrane bilayer systems we expanded our cholesterolcontaining lipid bilayer tests and analyzed several membrane biophysical properties, such as area per lipid (APL), membrane thickness and acyl chain order for DPPC and POPC membranes containing increasing concentrations of cholesterol at 323 K. Increasing concentrations of cholesterol induced both an increase in membrane thickness and a simultaneous decrease in APL in both PC bilayers (Figure , Supplementary Figure ). Regarding the magnitude of the cholesterolinduced membrane thickening (Figure ), neutron diffraction experiments have recovered headgroup peak-to-peak distances of 44.8 Å for DPPC bilayers containing 30% cholesterol at 300 K 107 , a 5.6 Å increase from pure DPPC bilayers. Running this particular system (Supplementary figure ), we recover an increase of 3.15 and 0.61 Å for the Martini 3 and Martini 2 systems, respectively. This corresponds to a 7.46 and 1.26 % increase relative to pure lipid bilayers. Compared to the 14 % increase observed experimentally, the Martini models show insufficient thickening. However, it is clear that the new Martini 3 cholesterol model shows improved behavior compared to the previous Martini 2 iteration. AA MD simulations of POPC bilayers containing cholesterol also observed an increase in phosphate-to-phosphate thickness from 37.1 Å to 40.4 Å with the addition of 20% cholesterol at 338 K 108 , compared to the 1.19 and 1.16 Å increase from our Martini 2 and Martini 3 simulations, respectively (Supplementary figure ). In this case, both models similarly underestimate the thickening effect. While Martini 3 correctly follows the trends in bilayer thickness, we systematically underestimate the thickening effect compared to both AA MD and experimental results. This was already the case in Martini 2 (Figure ). Still, Martini 3 performs better than Martini 2 in this aspect (especially for POPC), showcasing the improvements in both lipid and cholesterol models. This is particularly clear when looking at the percentual increase in thickness relative to pure lipid bilayers (Supplementary Figure ), which shows that the Martini 3 model consistently yields a larger increase in thickness compared to Martini 2. It is also worth noting that a recent comparison of AA cholesterol-POPC models found an overall overestimation of the cholesterol thickening effect upon the addition of over 20% cholesterol 109 . This observation hints that the underestimation of cholesterol-induced thickening by the Martini models relative to AA references may not be as big of an issue as previously thought. Nonetheless, ongoing developments focused on the lipid topologies and on the detail by which we represent acyl chains have the potential to further improve this feature.