The duration of such simulations is an obvious quantity that affects the predicted ABFEs. To systematically study its effect on the accuracy and precision of our predictions, we extended the length of each replica within the ensemble-simulation to 100 ns for a subset of 73 complexes (for the EQ approach) and to 50 ns for a subset of 81 complexes (for the NEQ approach). Table compares RMSE values as well as ∆G prot conf between replica lengths 10 ns and 100/50 ns for all these complexes system-wise. Figure displays a comparison between results from the first and the last 10 ns of the total 50 ns long replicas in case of NEQ calculations for 81 complexes systemwise. It is apparent that the effect of replica length is system-dependent. However, a closer look at the data reveals some interesting patterns. In all systems, the extent of change in raw RMSE and ∆G prot conf values (between 10 ns and 50/100 ns) are correlated such that a higher/lower change in raw RMSE is accompanied by a change of similar magnitude in ∆G prot conf . This is true for both EQ and NEQ approaches. This means that with longer simulation duration, conformational sampling of both holo and apo states may take place, improving the overall accuracy of predictions. However, this phenomenon is system-dependent and may not happen at all or only to a small extent for some systems even at 100/50 ns (such as Bromosporine, CDK2, TYK2, BRD4-2). This is evident from the large differences between RMSEs before and after adjustment for various systems using the replica length of 100 ns (2.26 kcal/mol for CDK2 and 1.06 kcal/mol for bromosporine) in case of the EQ approach and 50 ns (2.61 kcal/mol for BRD4-2, 6.61 kcal/mol for ROS1, 6.41 kcal/mol for TRKA and 6.71 kcal/mol for TYK2) in case of the NEQ approach. This is in agreement with a previous study where the authors studied two systems using extended simulations (1 µs long) to report that the accuracy of ABFEs improved only for one system, whereas it deteriorated for the other . Furthermore, RMSEs after adjustment (denoted as "RMSE adj ") are very similar at 10 ns and 100 ns in all cases (the largest difference being 0.43 kcal/mol in case of BRD4-1). This implies that a simple adjustment using an empirical correction term ∆G prot conf yields the benefits of extended simulation duration in most cases (with the obvious caveat that reference data is necessary to perform such a correction). Therefore, it is not desirable to run long duration simulations in general for ABFE calculations if we are only interested in the relative ranking of compounds. With regard to the optimal replica length in such calculations, we recommend 10 ns per replica for equilibrium simulations (applicable to both EQ and NEQ approaches) based on our results in Figure where we display the running averages of ∆G com for all 73 complexes up to 100 ns replica length for each system separately. It is manifest that in most cases, the ∆G com value either converges or changes only very slightly after 10 ns. Nevertheless, we would like to emphasise that all these observations are only relevant when one employs ensemble simulations (all our results in Table and Figures S9 and S10