BACKGROUND:Many patients visiting physiotherapists for musculoskeletal disorders face psychosocial challenges which may form a large barrier to recover. There are only a limited number of evidence based psychosocial therapies, but they are mainly based on breathing exercises. OBJECTIVE: to study which respiration frequency would lead to the highest relaxation, reflected in vagal tone derived from the heart rate variability (HRV) in healthy subjects. METHODS: A randomized controlled cross sectional study was performed. Respiration cycles of four, five, six, seven and eight breaths per minute (BPM) were delivered in randomized order for two minutes each. HRV metrics were measured during the sessions with electrocardiogram (ECG). Repeated Measures ANOVA’s were performed to analyze differences between breathing frequencies. RESULTS: 100 healthy volunteers were included (40 male). Standard Deviation of inter beat intervals (SDNN) values were significantly highest at 5 BPM, whereas the Root Mean Square of Successive Differences (RMSSD) values appeared highest at 7 breaths per minute (p< 0.01). High Frequency (HF) power was lowest at 4 BPM, whereas Low Frequency (LF) power was not significantly influenced by respiration frequency. CONCLUSIONS: Breathing at a frequency of 5 to 7 breaths per minute leads to highest HRV values, but there is no single respiration ratio that maximizes all metrics. Physiotherapists may use five to seven BPM as guidance to determine ideal breathing frequencies