Night-shift workers may develop poor cardiovascular health. Studies about heart rate variability (HRV) metrics could identify risk factors in this population and be used to examine the effectiveness of interventions for optimizing the health of night-shift workers. The purpose of this review was to examine the use of HRV methodologies in studies about night-shift work. Overall, 34 articles met the selection criteria and underwent a methodological critique. The main conclusion across these studies was that night-shift work could increase the sympathetic influences on the variability between heartbeats. In many cases, however, important methodological details were omitted (e.g., the number and duration of electrocardiogram recordings, sampling rates, R–R segment duration, wavelet transform methods). Recommendations include adding measures of disease outcomes, using ≥250 Hz sampling rates and 600-s R–R segments, and measuring sleep and circadian rhythms. With these approaches, researchers can design investigations that identify therapeutic targets for improving the health of night-shift workers. Int J Occup Med Environ Health. 2020;33(4):409–25