ObjectiveTo examine the effect of 24‐h shift work on autonomic nervous system function via heart rate variability (HRV) methodologies.MethodsElectronic databases (indexed in either PubMed, MEDLINE, CINAHL, SPORTDiscus, or OpenDissertations) were searched from January 1964 to March 2023. A modified Downs and Black checklist was used for assessing methodological quality and the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) approach was used to evaluate the quality of evidence. Study design, study population, study sample, shift work description, and assessment of HRV metrics and methods were extracted from each study.FindingsA total of 58 478 study articles were identified, of which 12 articles met inclusion criteria. Sample sizes varied from eight to 60 participants, with the ratio of low‐ to high‐frequency HRV (LF/HF) as the most common frequency‐domain variable reported. Of the nine included studies that observed LF/HF, three (33.3%) demonstrated a significant increase after 24‐h shift work. Moreover, of the five studies that reported HF, two (40%) noted a significant decrease after 24‐h shift work. When observing risk of bias, two (16.6%) studies were low quality, five (41.7%) were moderate quality, and five (41.7%) were high quality.InterpretationThere were inconsistent findings demonstrating an effect of 24‐h shift work on autonomic function, with a suggested shift away from parasympathetic dominance. Discrepancies in HRV methodologies, such as the duration of recordings and hardware used for measurement, may have contributed to the disparity in findings. In addition, differences in roles and responsibilities across occupations may explain the incongruence in findings across studies.