While the primary goal of introducing buffers is to avoid the artifacts associated with a non-neutral periodic simulation box, there can be other artifacts as well. In particular, the undersolvation caused by solvent orientational constraints due to periodic conditions could lead to finite-size effects especially for small boxes. To understand if such finite-size effects affect the results of constant pH simulations, we investigated how the distribution of the λ-coordinates depends on the system size. We, therefore, performed constant pH MD simulations for three different box sizes and at various ionic strengths. All simulations were performed at pH=pK a and without a barrier in the biasing potential. The uniformity of the distributions in these simulations, shown in Figure ), suggests that for the box sizes tested, the finite-size effects are negligible.