The contribution of carbon and nitrogen reserves to regrowth following shoot removal and the processes involved in the reduction of nodule functioning were studied in alfalfa plants (Medicago sativa L.). To do so, isotopic labelling was conducted at root and canopy level with both 15N2 and 13C-depleted CO2 on exclusively nitrogen fixing alfalfa plants. Our results indicate that the roots were the main sink organs before shoot removal as expected. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules in order to sustain respiratory activity. However, this carbon and nitrogen derived from the root did not overcome carbon and nitrogen limitation in nodules and leaves. Together with the limited carbohydrate availability, the up-regulation of nodule peroxidases indicates the involvement of oxidative stress in a worse nodule performance. Fourteen days after shoot removal, leaf and nodule performance were completely reestablished.