In middle-aged persons, moderate physical activity energy expenditure (PAEE) has been shown to be associated with increased vagal-related heart rate variability (HRV) indexes and better health status. The purpose of this study was to determine whether a very high PAEE has greater effect on vagal-related HRV indexes and self-estimates of well-being in middle-aged subjects having distinct long-term physical activity profiles. Forty-four subjects were divided into three groups on the basis of the sport score of the Modified Baecke Questionnaire for Older Adults: sedentary (SED, n = 15), moderately-trained (MT, n = 16), and highly-trained subjects (HT, n = 13). PAEE was estimated by triaxial accelerometry during one week. Time and frequency domain HRV indexes were determined during quiet periods in the morning on 5-min R-R interval segments under controlled breathing. Quality of life was evaluated using the SF-36 health survey questionnaire. PAEE was significantly different for each group (374.5 +/- 13.8, 616.8 +/- 22.4, and 1086.6 +/- 43.2 kcal . day (-1) for SED, MT, and HT, respectively, p < 0.001). MT presented significantly higher vagal-related HRV indexes than SED and HT (p < 0.05). None of the HRV indexes was significantly greater in HT than in SED. MT and HT had similar health status scores, which were significantly higher than for SED. These results indicate that in middle-aged subjects, habitual moderate PAEE is associated with greater vagal tone and self-estimates of well-being compared to low PAEE. In contrast, very high PAEE is associated with similar vagal-related indexes as low PAEE, despite better overall health status.