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The contribution of carbon and nitrogen reserves to regrowth following shoot removal has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both 15N2 and 13C-depleted CO2 on exclusively nitrogen-fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root-derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N2-fixing machinery, availability of Cnew and Nnew strongly diminished in the plants due to their replacement by C and N assimilated during the post-labelling period. In summary, our study indicated that during the first week of regrowth, root-derived C and N remobilization did not overcome C- and N-limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re-established.