Nine participants were included in this double-blind, crossover, placebo-controlled study evaluating the pharmacology of two consecutive doses of ayahuasca in humans. Participants were administered a combination of ayahuasca containing 0.75 mg/kg N,N-dimethyltryptamine (DMT) and placebo conditions [placebo plus placebo, ayahuasca (Aya 0) plus placebo, ayahuasca (Aya 1) plus ayahuasca (Aya 2)] in three experimental sessions with 4 hours between conditions and a washout period of at least one week between sessions. Subjective drug effects were measured using a visual analog scales (VAS), the Hallucinogen Rating Scale (HRS), and the Addiction Research Center Inventory (ARCI). Neurophysiological effects were measured using EEG. Cardiovascular and autonomic effects were measured using blood pressure, heart rate, body temperature, and pupillary diameter. Lymphocyte subpopulations were quantified from blood samples, as was serum growth hormone (GH) and prolactin concentrations which were determined by a chemiluminescence immunoassay system. After two consecutive doses of ayahuasca doses, DMT plasma concentrations, scores in subjective and neurophysiological variables, and serum prolactin and cortisol were found to be significantly higher. A second dose of ayahuasca reduced systolic blood pressure and heart rate, and significantly decreased levels of growth hormone (GH).