Heart rate variability (HRV) refers to the beat-to-beat variation of the cardiac cycle. Since heart rate is modulated on a beat-to-beat basis by the combined influence of the sympathetic and parasympathetic nervous system at the sinus node level, HRV has been considered an indirect biomarker of cardiac autonomic control and widely exploited for the assessment of autonomic function in many pathological subjects. This focus article summarizes the main findings derived from HRV analysis applied to 24‑h Holter monitoring in both cardiac and non-cardiac diseases as well as in physiological conditions in the healthy population. Even if the prognostic role of HRV indices is well recognized and its use ever more widespread, its implementation in the diagnostic and prognostic processes in routine clinical practice remains limited. Several reasons for these limitations can be identified: first the lack of reliable reference values, and secondly, the low specificity of HRV indices in particular when considering the constant evolution of clinical practice and therapeutic approaches, making it difficult to refer to a specific and stable combination of clinical and HRV markers. Therefore, the clinical use of HRV should be further investigated. Finally, HRV represents a substantial tool for investigating the physiological conditions in healthy people that can have important implications in primary prevention and the understanding of gender differences, as well as in sport and occupational medicine.