In the NEQ approach, the overlap of the work distributions was very poor, even with 10 ns/transition (SI Figure ). Running 10 ns per transition requires considerable computational expense and removes much of the benefit of the NEQ approach. Given the same total simulation time for different NEQ protocols, the lengths of the switching transitions had a huge impact on the estimated free energy change and different protocols only converged to the same result within large uncertainty and after long simulation times. This suggests that the uncertainty of the ∆G estimates is larger when work distributions lack overlap.