While performing a single repeat of US along a one-dimensional coordinate has emerged as the established approach for computing PMFs, our results suggest that this may lead to an incorrect estimation of binding affinity, as there can be other pathways that lead to different PMFs. To estimate this effect, multiple repeats are needed. However, when the differences between PMFs from multiple repeats are large, obtaining meaningful results is challenging. While averaging PMF profiles is one possibility, as introduced by Niskikawa et al., 102 this would assume equal weights for all paths sampled in the repeats. Because of the differences between the PMFs computed for multiple repeats of US along the same 1D reaction coordinate, we assume that not all non-equilibrium work has dissipated in our simulations. Therefore, computing the average with equal weights will also overestimate the free energy difference since the total work exceeds the free energy: 103 W ≥ ∆G with W the total work performed, and ∆G the free energy difference between the states.