Simulations with the generated λ-schedules showed no visible reduction in the overall standard error of the mean of the free energy change for equivalent sampling time compared to the "manually-optimised" protocol (Figure ); rather, all schedules other than that with 4.0 kcal mol -1 speed showed similar inter-run uncertainties. This is as expected given that the protocol did not account for differences in statistical inefficiency between stages. This may have also been anticipated from the results of Nguyen and Minh, who found that given sufficient overlap, the uncertainty collapsed purely as a function of total sampling time. 130 However, the 4.0 kcal mol -1 thermodynamic speed protocol often showed substantially greater uncertainty, as anticipated from the negligible overlap.