The potential of a plant species to acquire nutrients depends on its ability to explore the soil by its root system. Co-cultivation of different species is anticipated to lead to vertical root niche differentiation and thus to higher soil nutrient depletion. Using a qPCR-based method we quantified root biomass distribution of four catch crop species in vertical soil profiles in pure vs. mixed stands. Pure stands of mustard and phacelia robustly reached 70 cm soil depth, while oat preferably colonized upper soil layers, and clover developed the shallowest and smallest root system. Analysis of residual nitrate pools in different soil depths and correlation with root biomass showed that, besides rooting depth also root biomass determines soil nitrogen depletion. While occupying the same vertical niches as in pure stands, mustard and phacelia dominated total root biomass of the mix. In contrast, root biomass of clover and oat was severely suppressed in presence of the other species. Below-ground biomass profiling indicated low niche complementarity among the root systems of the examined species. Nonetheless, the mixture mostly overyielded root biomass of the pure stands and thus shows higher potential for efficient soil exploration by roots.