BACKGROUND: Reduced heart rate variability (HRV), a measure of cardiac autonomic dysfunction, is a risk factor for coronary artery disease. Diet can influence HRV, but this association may be confounded by genetic and environmental factors. METHODS AND RESULTS: We administered the Willett Food Frequency Questionnaire to 276 middle-aged male twins. We derived a score measuring the extent to which an individual\'s diet conformed to the Mediterranean diet following a published algorithm. The higher the score, the greater the similarity to the Mediterranean diet. All twins underwent 24-hour ambulatory ECG recording. Time and frequency domain measures of HRV were calculated. Mixed-effects regression was used to partition the association into between- and within-twin pair differences. After adjusting for energy intake, other nutritional factors, shared genes, and common environment, a 1-unit higher score was significantly associated with 3.9% to 13% higher time and frequency domain HRV parameters. Further controlling for known cardiovascular risk factors and use of fish oil supplements and medications did not substantially change the estimates. CONCLUSIONS: The Mediterranean dietary pattern is associated with higher HRV.