Heart rate variability (HRV) is an analysis of milliseconds variations in intervals between heartbeats and has become an increasingly used tool for clinical investigation of fatigue, especially in athletes. Eliciting an indirect index of the autonomic nervous system regulation on the heart rate, HRV correlates with different fatigue states and appears to be a powerful biomarker in their monitoring. This article presents the tools to familiarize with this method while detailing good practices for use and interpretation. A method allowing characterization of different fatigue states is also presented for a clinical use with a systemic approach.