Background <p>Metabolomics describes the metabolic profile of an organism at a given time by the concentrations of its constituent metabolites. When studied over time, metabolite concentrations can help understand the dynamical evolution of a biological process. However, metabolites are involved into sequences of chemical reactions, called metabolic pathways, related to a given biological function. Accounting for these pathways into statistical methods for metabolomic data is thus a relevant way to directly express results in terms of biological functions and to increase their interpretability.</p> Methods <p>We propose a new method, phoenics, to perform differential analysis for longitudinal metabolomic data at the pathway level. In short, phoenics proceeds in two steps: First, the matrix of metabolite quantifications is transformed by a dimension reduction approach accounting for pathway information. Then, a mixed linear model is fitted on the transformed data.</p> Results <p>This method was applied to semi-synthetic NMR data and two real NMR datasets assessing the effects of antibiotics and irritable bowel syndrome on feces. Results showed that phoenics properly controls the Type I error rate and has a better ability to detect differential metabolic pathways and to extract new impacted biological functions than alternative methods. The method is implemented in the <Emphasis FontCategory="SansSerif">R</Emphasis> package phoenics available on CRAN</p>