Background: Previous studies have suggested that individuals recovered from anorexia nervosa (AN) are characterized by increased serotonergic (5-HT) activity that might be related to elevated levels of anxiety. Assuming these traits to be also present in individuals at risk for AN, it was further hypothesized that restricting food intake might be a means to temporarily alleviate dysphoric affective states by reducing central nervous availability of tryptophan (TRP), the sole precursor of 5-HT. One study that supported this hypothesis found anxiolytic effects in individuals with a history of AN during an experimentally induced short-term depletion of TRP supply to the brain. Methods: In this placebo-controlled, double-blind cross-over study, 22 patients weight-recovered from AN (recAN) and 25 healthy control participants (HC) completed questionnaires assessing anxiety and momentary mood during acute tryptophan depletion (ATD), a dietary intervention that lowers central 5-HT synthesis. Results: The ATD procedure effectively reduced the ratio of TRP to competing for large neutral amino acids in the peripheral blood, indicating decreased TRP supply to the brain. Effects of ATD on anxiety and mood did not differ between recAN and HC. Bayesian null hypothesis testing confirmed these initial results. Discussion: Our results do not support the hypothesis that short-term depletion of TRP and its impact on the brain 5-HT reduces anxiety or improves mood in AN. As the evidence for the role of 5-HT dysfunction on affective processes in patients with AN is limited, further studies are needed to assess its relevance in the pathophysiology of AN.