Algal cells are known for improved accumulation of long chain fatty acids when grown in continuous flow cultures under conditions where nutrients, such as nitrogen and/or phosphorous, are limited. Food waste has been shown to be an appropriate source of carbon, nitrogen and phosphorous compounds to be used as nutrients in heterotrophic microalgal biomass production. Food waste, however, has unpredictable contents of the three mentioned compounds, which makes an application in continuous flow cultures where defined concentrations of nutrients are required challenging. Therefore, this study aimed on developing continuous flow cultures of the heterotrophic microalga Chlorella pyrenoidosa solely based on mixed restaurant food waste for improved fatty acid production. Food waste was enzymatically digested using amylolytic or proteolytic enzymes, and the obtained hydrolysate was used for the cultivation of C. pyrenoidosa in continuous flow cultures under nitrogen and/or phosphate limitation. In a nutrient sufficient batch culture, C. pyrenoidosa contained 103.8 mg g⁻¹ lipids, while up to three times more lipids was found in biomass cultured under nutrient limited conditions. C. pyrenoidosa also accumulated up to 619.0 mg g⁻¹carbohydrates. With decreasing dilution rate and increasing nutrient stress, the carbohydrate content could be decreased to 390.9 mg g⁻¹. The outcomes of this study extended our understanding on how to utilize complex media in continuous flow cultures of algal cells which are carried out under specific nutrient limitation for improved fatty acid accumulation.