The aim of this work was to study the stimulation of lipid production on the microalga Ankistrodesmus falcatus by varying cultivation conditions during the stationary phase. The effect of three factors (presence and absence of nitrogen, phosphorus, and light) has been tested once the cultures reached the stationary phase with the aim to increase the value of the biomass for further applications. Lipid content, elemental composition, Nile red fluorescence evolution, and calorific value of microalgal biomass were studied as well as biomass growth. Biomass presented a lipid content of 36.54 % at the end of the first stage, while at the end of the second stage, the experiments with the absence of phosphorus increased their lipid content until 45.94 and 44.55 %, the first with nitrogen and light presence and the second with absence of all factors. The combination of phosphorus absence and nitrogen and light presence achieved the highest lipid productivity (20.27 mg/L/day). The two-stage strategy to culture microalgae is a feasible option to increase the economic or energetic value of biomass.