Burnout is a work-related mental health impairment comprising three dimensions: emotional exhaustion, depersonalization, and reduced personal accomplishment. Reiki aims to help replenish and rebalance the body's energetic system, thus stimulating the healing process. The objective of this placebo-controlled, repeated measures, crossover, single-blind, randomized trial was to analyze the immediate effects of Reiki on heart rate variability (HRV), body temperature, and salivary flow rate and cortisol level in health care professionals with burnout syndrome (BS). Participants included 21 health care professionals with BS, who were asked to complete two visits to the laboratory with a 1-week interval between sessions. They were randomly assigned the order in which they would receive a Reiki session applied by an experienced therapist and a placebo treatment applied by a therapist with no knowledge of Reiki, who mimicked the Reiki treatment. Temperature, Holter ECG recordings (standard deviation of the normal-to-normal interval [SDNN], square root of mean squared differences of successive NN intervals [RMSSD], HRV index, low frequency component [LF], and high frequency component [HF]), salivary flow rate and cortisol levels were measured at baseline and postintervention by an assessor blinded to allocation group. SDNN and body temperature were significantly higher after the Reiki treatment than after the placebo. LF was significantly lower after the Reiki treatment. The decrease in the LF domain was associated with the increase in body temperature. These results suggest that Reiki has an effect on the parasympathetic nervous system when applied to health care professionals with BS.