Oleaginous diatoms, including the marine model species Phaeodactylum tricornutum, have been identified as promising sources of compounds with a biotechnological interest including triacylglycerol and the soluble polysaccharide chrysolaminarin. These two molecules are accumulated in cells under nutrient starvation conditions, however, their consumption after nutrient replenishment lack a precise characterization. In this study, two ecotypes of P. tricornutum (Pt1 and Pt4) have been subjected to a nitrate starvation followed by a resupply to monitor their response through biochemical assays and gene expression quantification. We highlighted that both ecotypes experienced a two-step response to nitrogen resupply, with a rapid initial consumption of stored soluble carbohydrates probably allowing the restart of photosynthesis and protein synthesis, followed by a consumption of neutral lipids fueling cell division. Some genes were particularly upregulated after resupply, especially those encoding the lipases Phatr3_EG02408 and Phatr3_EG00720 and the exoglucosidase Phatr3_J43302, suggesting their role in degradation processes. Additionally, ecotype Pt4 recovered faster from stress than ecotype Pt1, possibly due to differences in specific gene regulations. Overall, these findings provide potential targets for understanding lipid and carbohydrate degradation, and for creating high-lipid producing strains.