For all other systems, any initial transients were substantially smaller and this method discarded no time to equilibration. Inspection of the equilibration times for all stages showed that the paired t-test method rarely discards any data when there is no visible initial transient (Figure ). This is in contrast to the popular method of Chodera which often discards a substantial amount of data even when there is no obvious initial transient, even when it is applied to the mean trace (see MDM2-Nutlin bound vanish in Figure , T4L bound restrain in Figure ). We compared the absolute differences between the ∆G estimates obtained with both equilibration detection methods to the final 30 ns non-adaptive bound vanish results (Figure ). In every case, the difference is smaller for the paired t-test, which could be due to reduced variance arising from retaining more of the data. However, we have a small sample size of 5, and there is no evidence for a significant difference between the two methods at 95 % confidence based on the Wilcoxon signed-rank test (p=0.06).