Microalgae are one of the most promising biodiesel feedstocks due to their efficiency in CO2 fixation and high neutral lipid productivity. Nutrient-stress conditions, including nitrogen starvation, enhance neutral lipid content, but at the same time lead to a reduction of biomass. To maximize lipid production in the diatom Skeletonema marinoi, we investigated two different nitrogen starvation approaches. In the first experimental approach, inocula were effectuated in modified f/2 media with decreasing nitrogen concentration, while in the second experiment, nitrate concentration was gradually reduced through a collection/resuspension system in which the culture was periodically collected and resuspended in culture medium with a lower nitrate concentration. In the first approach, the neutral lipid accumulation was accompanied by a strong biomass reduction, as was expected, whereas the second experiment generated cultures with significantly higher neutral lipid content without affecting biomass production. The total proteins and total carbohydrates, which were also quantified in both experiments, suggest that in S. marinoi, neutral lipid accumulation during nutrient starvation did not derive from a new carbon partition of accumulated carbohydrates.