This updated guideline replaces the “Guideline for the application of heart rate and heart rate variability in occupational medicine and occupational health science” first published in 2014. Based on the older version of the guideline, the authors have reviewed and evaluated the findings on the use of heart rate (HR) and heart rate variability (HRV) that have been published in the meantime and incorporated them into a new version of this guideline. This guideline was developed for application in clinical practice and research purposes in the fields of occupational medicine and occupational science to complement evaluation procedures with respect to exposure and risk assessment at the workplace by the use of objective physiological workload indicators. In addition, HRV is also suitable for assessing the state of health and for monitoring the progress of illnesses and preventive medical measures. It gives an overview of factors influencing the regulation of the HR and HRV at rest and during work. It further illustrates methods for measuring and analyzing these parameters under standardized laboratory and real workload conditions, areas of application as well as the quality control procedures to be followed during the recording and evaluation of HR and HRV.