To inform the development of our adaptive protocols, we first performed non-adaptive ABFE calculations on five protein-ligand systems (Figure ). These were intended to span a range of ligand sizes, rates of convergence, and sampling challenges. Benzene/T4L is a common test system for ABFE methodologies; 7,117 MIF/MIF180 is subject to slow rehydration of the binding site upon ligand decoupling; 103,118 the MDM2 complexes were used to develop the group's previous adaptive protocol; and the slow convergence of the PDE2a complex was highlighted by Huggins. We ran 5 independent replicate runs for each system, all using the manually-optimised λ schedule from our previous work. Firstly, we investigated short-timescale behaviour by running all windows for a total of 0.2 ns and using all data for analysis. We then carried out a more realistic protocol optimised for MIF/MIF180 in our previous work -all windows were run for 6 ns with the initial 1 ns discarded to equilibration, other than the bound vanish stages, which were run for 8 ns with the initial 3 ns discarded to equilibration. Finally, we investigated longer timescale behaviour by running all windows for 30 ns and discarding the first 10 ns of data to equilibration. The protocols are referred to as 0.2 ns, 6 ns, and 30 ns. As we aimed to compare between protocols rather than with experiment, we tolerated two potential sources of error which we may not have otherwise;