The effect of the degree of isotopic substitution of the aqueous medium on the adsorption kinetics and the surface dilational rheological behavior at the water/air interface of the globular protein β-lactoglobulin was investigated. Aqueous solutions with fixed concentrations of 1 μM protein and 10 mM hydrogenous buffer with controlled pH 7 were prepared in H2O, D2O, and an isotopic mixture of 8.1% v/v D2O in H2O (called air contrast matched water, ACMW). Using a bubble shape analysis tensiometer, we obtained various experimental dependencies of the dilational viscoelasticity modulus E as a function of the dynamic surface pressure and of the frequency and amplitude of bubble surface area oscillations, either in the course of adsorption or after having reached a steady state. In general, the results revealed virtually no effect from substituting H2O by ACMW but distinct albeit relatively weak effects for intermediate adsorption times for D2O as the aqueous phase. In the final stage of adsorption, established after around 10 h, the equilibrium adsorption and the dilational rheological behavior of all protein layers under investigation are only very weakly affected by the presence of D2O. The obtained results help to design experimental protocols for protein adsorption studies, for example, by neutron reflectivity.