The recent progress of Atom Probe Tomography (APT) has opened up atomic-scale elemental analysis including hydrogen species. For APT measurements, the use of deuterium is highly recommended, due to its low mobility compared to the fast and quantum mechanically tunneling isotope hydrogen. In addition, deuterium can be distinguished from hydrogen originating from the APT analysis chamber. To date, however, APT studies on materials with high D concentrations are scarce. In this study, the D concentration profile in a Fe/V multi-layered film sample was investigated, and spanned a wide concentration range. The mean hydrogen isotope concentration was alternatively quantified by electromotive force (EMF) measurements on a similar Fe/V film, thus verifying the APT results. The reduction found in the D concentration at the Fe/V interface results from local alloying at the Fe/V interfaces which accompanies a change in the available volume in the V lattice. Even at the same Fe concentration, the shape of the observed D depth profile was asymmetric at high D2 pressures. This indicates a stress impact caused by the deposition sequence.