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Background Spectral analysis of Heart Rate Variability (HRV) is a simple, non-invasive technique that is widely used in sport to assess sympatho-vagal regulation of the heart. Its employment is increasing partly due to the rising usage of wearable devices. However data acquisition using these devices may be suboptimal because they cannot discriminate between sinus and non-sinus beats and do not record any data regarding respiratory frequency. This information is mandatory for a correct clinical interpretation. Methods This study involved 974 elite athletes, all of them underwent a complete autonomic assessment, by way of Autoregressive HRV analysis. Results In 91 subjects (9% of the total population) we observed criticalities of either cardiac rhythm or respiration. Through perusal of one-lead ECG analysis we observed that 77 subjects had atrial or ventricular ectopy, i.e. conditions which impair stationarity and sinus rhythm. Running anyway autonomic nervous system analysis in this population, we observed that RR variance and raw values of LF and HF regions are significantly higher in arrhythmic subjects. In addition 14 subjects had slow (about 6 breath/min, 0.1 Hz) respiration. This condition clouds the separation between LF from HF spectral regions of RR interval variability, respectively markers of the prevalent sympathetic and vagal modulation of SA node and of their synergistic interaction. Conclusions Caution must be payed when assessing HRV with non-ECG wearable devices. Recording ECG signal and ensuring that respiratory rate is higher than 10 breath/min are both prerequisites for a more reliable analysis of HRV particularly in athletes. [ABSTRACT FROM AUTHOR]