D2O, an isotope of H2O, is commonly used as a solvent in neutron scattering; the large difference in scattering length density between H and D can provide better contrast between the sample and the solvent. However, this is of concern for studies using enzymes as the use of D2O can influence protein interactions (due to differences in hydrogen bonding) and is therefore expected to affect the function, activity and solubility of enzymes. Neutron-based in vitro digestion assays on proteins, including those found in food or as oral protein and peptide drugs, often involve different solvents or pH conditions where the activity of the digestive enzyme may not be optimal. Herein, we investigate the structure and activity of the main gastric protease, porcine pepsin, in both H2O and D2O at pH values in the range 1 – 8. We showed that the activity of pepsin was lower in D2O, although the relative change in activity with pH was similar for both solvents. We demonstrated using a combination of SAXS and CD that this relative change in activity was not related to any structural change within the protein but was, rather, linked to relative changes in solubility of the protein.