values at 4 fs. In the aqueous phase, for instance, all λ states show significant deviations at 4 fs. In the complex phase, although some λ states at 4 fs do not show statistically significant differences, likely due to larger standard deviations in the data, a similar trend of decreasing p-values with increasing timestep is observed. This pattern is consistent with the previously discussed observations for the cAMP and ethane systems, reinforcing the conclusion that larger timesteps, particularly 4 fs, can introduce significant inaccuracies in the calculation of dU dλ , regardless of the specific system or phase. The t-test results again emphasize the preference for smaller timesteps to ensure convergence and reliability in thermodynamic integration calculations.