Heart rate variability (HRV) analysis is a widely used technique to assess sympatho-vagal regulation in response to various internal or external stressors. However, HRV measurements under free-moving conditions are highly susceptible to subjects\' physical activity levels because physical activity alters energy metabolism, which inevitably modulates the cardiorespiratory system and thereby changes the sympatho-vagal balance, regardless of stressors. Thus, researchers must simultaneously quantify the effect of physical activity on HRV to reliably assess sympatho-vagal balance under free-moving conditions. In the present study, dynamic body acceleration (DBA), which was developed in the field of animal ecology as a quantitative proxy for activity-specific energy expenditure, was used as a factor to correct for physical activity when evaluating HRV in freely moving subjects. Body acceleration and heart inter-beat intervals were simultaneously measured in cattle and sheep, and the vectorial DBA and HRV parameters were evaluated at 5-min intervals. Next, the effects of DBA on the HRV parameters were statistically analyzed. The heart rate (HR) and most of the HRV parameters were affected by DBA in both animal species, and the inclusion of the effect of DBA in the HRV analysis greatly influenced the frequency domain and nonlinear HRV parameters. By removing the effect of physical activity quantified using DBA, we could fairly compare the stress levels of animals with different physical activity levels under different management conditions. Moreover, we analyzed and compared the HRV parameters before and after correcting for the mean HR, with and without inclusion of DBA. The results were somewhat unexpected, as the effect of DBA was a highly significant source of HRV also in parameters corrected for mean HR. In conclusion, the inclusion of DBA as a physical activity index is a simple and useful method for correcting the activity-specific component of HRV under free-moving conditions.