The results reveal a notable consistency in dU dλ values across timesteps of 0.5, 1, and 2 fs. The average dU dλ values within this range remain relatively stable, varying between approximately 0.02 and 0.50 kcal/mol. However, a significant increase in the magnitude of dU dλ is observed when comparing the 0.5 fs timestep to the 4 fs timestep. The differences between the average dU dλ values obtained with 4 fs and the ones with 0.5 fs can reach up to 3 kcal/mol in the all SC case of the cAMP system, indicating a potential deviation in the calculated thermodynamic properties at this larger timestep. This trend suggests that while smaller timesteps provide more consistent results, larger timesteps may introduce inaccuracies in the estimation of dU dλ . A similar pattern of increasing dU dλ with increasing timestep was also observed during the ethane to ethanol transformation.