Elastic incoherent neutron scattering was used to explore solvent isotope effects on average macromolecular dynamics in vivo. Measurements were performed on living E. coli bacteria containing H2O and D2O, respectively, close to physiological conditions of temperature. Global macromolecular flexibility, expressed as mean square fluctuation (MSF) values, and structural resilience in a free energy potential, expressed as a mean effective force constant, hk0i, were extracted in the two solvent conditions. They referred to the average contribution of all macromolecules inside the cell, mostly dominated by the internal motions of the protein fraction. Flexibility and resilience were both found to be smaller in D2O than in H2O. A difference was expected because the driving forces behind macromolecular stabilization and dynamics are different in H2O and D2O. In D2O, the hydrophobic effect is known to be stronger than in H2O: it favours the burial of non-polar surfaces as well as their van der Waals’ packing in the macromolecule cores. This may lead to the observed smaller MSF values. In contrast, in H2O, macromolecules would present more water-exposed surfaces, which would give rise to larger MSF values, in particular at the macromolecular surface. The smaller value suggested a larger entropy content in the D2O case due to increased sampling of macromolecular conformational substates.