1H nuclear magnetic resonance (NMR)-based metabolomics has been applied to study the compatible interaction between tomato plants and Tomato Mosaic Virus (ToMV). A detailed time course of metabolic fingerprinting of ToMV-inoculated and non-inoculated systemically infected tomato leaves has provided a fundamental understanding of the metabolic state of the plant not only in response to ToMV infection, but also under various physiological conditions. By this analytical platform a total of 32 metabolites including amino/organic acids, sugars, phenylpropanoids, flavonoids and other miscellaneous compounds were detected. Using multivariate data analysis, we have identified a subset of metabolites induced during the plant defence response and metabolites whose accumulation was dependent on the developmental stage, the position of the leaf on the stem, and the harvesting time. Specifically, a general time-dependent decrease in organic acids, amino acids (excluding asparagine), phenylpropanoids and rutin was observed in individual leaves. In addition, metabolite alterations were also found to correlate with the developmental stage of the leaf: high levels of organic acids, some amino acids, phenylpropanoids, and flavonoids were found in lower leaves while elevated amounts of sugars were present in the upper ones. Moreover, a marked variation in the content of some metabolites was also observed to be associated to the asymptomatic ToMV infection both in inoculated and systemically infected leaves. While flavonoids accumulated in virus-inoculated leaves, increased levels of phenylpropanoids were observed in non-inoculated leaves where ToMV actively replicates. Finally, diurnal changes in the metabolite content were also observed: an increase of amino acids and organic acids (except glutamic acid) were observed in the samples collected in the morning, whereas sugars and secondary metabolite levels increased in the tomato leaves harvested in the evening.