Different charging conditions aimed at introducing significant hydrogen concentrations without microstructural damages in a 316L austenitic stainless steel were investigated. The equivalent hydrogen pressure developed at the surface of the samples during cathodic charging was estimated from hydrogen concentration measurements. A clear hydrogen absorption, controlled by diffusion, was evidenced during the immersion of 316L steel samples in 30% MgCl 2 at the open circuit potential at 117 °C. Deuterium profiling by SIMS was performed to check the validity of the few literature data on hydrogen diffusivity in the near room temperature range in this material. On the other hand, the macroscopic effects of hydrogen on the tensile characteristics of the steel were investigated and compared at 20 °C and at −196 °C with samples cathodically pre-charged, charged during tensile straining or pre-charged at high temperature–high pressure in gas phase. Hydrogen is shown to affect both the short range and the long range forces exerted on the strain-induced mobile dislocations. The hydrogen-induced softening effect observed at 20 °C and the systematic decrease of the ductility support a mechanism involving the enhanced transport of hydrogen atoms by mobile dislocations. This mechanism is confirmed by the absence of softening and of ductility loss at −196 °C and by the strain-enhanced tritium desorption from samples cathodically pre-charged with tritium, measured by β counting during tensile deformation.