Rapid tryptophan depletion studies investigate serotonin using amino acid precursor depletion, which transiently reduces the brain level of serotonin. This study compares the effects of serotonin reduction given on the first test day (when the situation is novel) with the effects of serotonin reduction given on the second test day (when the environment and test battery are familiar). A total of 24 healthy young males were given either active tryptophan depletion or placebo in this randomized cross-over design, while impulsivity was measured by a continuous performance test. The participants showed more impulsive responses and reduced attention during tryptophan depletion, but only when this was given on the first test day when the task was novel. This could be caused by a synergic effect between novel situations and reduced neurotransmission of serotonin.