Root system senescence and nitrogen (N) release from red clover (Trifolium pratense L.) plants, grown under semi-sterile conditions and a controlled environment, were studied for 28 days following temporary or prolonged abiotic stress. Plants stressed temporarily, to simulate grazing, recovered with no additional N lost in leachate. In contrast, plants subjected to prolonged stress that simulated overwintering conditions and inhibited shoot re-growth survived stress lasting 7 days, but plant viability was reduced to 50% by 14 days and 0% at 21 days. There were no significant differences in root protein, catalase activity, root death index or total N loss in leachate over 21 days, but by 28 days total N loss in leachate increased to 214% above control levels, with a 433% increase in total oxidized N. This increase in N loss between 21 and 28 days indicated the start of cellular breakdown of the root system, coinciding with the failure of plants to recover. Key enzyme activities and protein concentrations in nodules decreased rapidly over 10 days’ prolonged stress. cDNA–amplified fragment length polymorphism (AFLP) analysis identified contaminating bacterial and fungal genes, along with plant gene sequences with consistent or altered expression profiles. Four plant sequences, glyceraldehyde-3-phosphate dehydrogenase (Tp-gapdh1), nodule senescence reduced (Tp-nsr1), nodule senescence enhanced (Tp-nse1) and a cysteine protease gene (Tp-cp8) were differentially expressed throughout the plant: Tp-nsr1 and Tp-nse1 have potential as molecular markers for nodule senescence. Root and nodule death in agricultural legumes, such as red clover, are implicated in N release into watercourses and the wider environment. Differences in the ability of these plants to survive prolonged stress lasting 14 days, and the delayed release of root N into leachate until 28 days after the stress, highlight the potential for the development of new red clover varieties with different rates of root system senescence.