Purpose: Trastuzumab, effective in about 15% of women with breast cancer, downregulates signalling through the Akt/PI3K and MAPK pathways. These pathways modulate glucose and phospholipid metabolism which can be monitored by [18F]FDG-PET and 31P-NMR spectroscopy respectively. Here the relationship between response of HER-2 overexpressing tumours and changes in [18F]-FDG incorporation and 31P-NMR detectable phosphomonesters were examined. Experimental: Xenografts derived from HER2-overexpressing MDA-MB-453 human breast tumour cells were grown in SCID mice, treated with trastuzumab for 15 days then [18F]-FDG uptake determined and 31P-NMR carried out on chemical extracts of the tumours. Western blots were carried out to determine protein expression of Hexokinase 2 and glut1. Results: [18F]-FDG incorporation, hexokinase II and glut1 protein expression and the concentration of phosphocholine and phosphoethanolamine in chemical extracts subjected to 31P-NMR were significantly decreased in the xenografts in the trastuzumab treated mice compared with xenografts from the PBS-injected group. Conclusions: Changes in FDG incorporation and 31P-NMR spectral changes can accompany response of HER2 expressing breast cancer xenografts to trastuzumab. This is the first study to show parallel changes in [18F]FDG and 31P-NMR detectable metabolites accompany response to targeted anti-cancer treatment.