Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. We characterized flag leaf senescence at 7, 14 and 21 days past anthesis (dpa) in two near-isogenic barley lines varying in the allelic state of the HvNAM1 transcription factor gene, which influences senescence timing. Metabolomics and microscopy indicated that, as senescence progressed, thylakoid lipids were transiently converted to neutral lipids accumulating in lipid droplets. Senescing leaves also exhibited an accumulation of sugars including glucose, while nitrogen compounds (nucleobases, nucleotides, and amino acids) decreased. RNA-Seq analysis suggested lipid catabolism via β-oxidation and the glyoxylate cycle, producing carbon skeletons and feeding respiration in replacement of diminished carbon supply from photosynthesis. Comparison of the two barley lines highlighted a more prominent upregulation of Heat Stress Transcription Factor (HSF) and chaperone-encoding genes in the late-senescing line, suggesting a role for these genes in the control of leaf longevity. While numerous genes with putative roles in nitrogen remobilization were upregulated in both lines, several peptidases, nucleases and nitrogen transporters were more highly induced in the early-senescing line; this finding identifies processes and specific candidates which may affect nitrogen remobilization from senescing barley leaves, downstream of the HvNAM1 transcription factor.