Heart rate variability (HRV) is a noninvasive physiological marker used to assess autonomic nervous function and can be recorded over the short or long term. Long-term recording is a good method for assessing mortality and patient prognosis, while short-term measurement is widely used due to practical advantages and reproducibility. However, little is known about whether a short-term assessment reflects the variation in the overall heart rate of workers. This study evaluated the relationship between the 24-hour and 5-minute HRV, which was selected from a 24-hour recording. The study population was 153 male workers at the National Rail Company in Korea, who had their heart rates assessed for 24 hours. In the time and frequency domains, the correlations of the HRV between 24 hours and 5 minutes were calculated for the entire time and limited times (09:00-17:00). We found modest correlations in the time (R = 0.614-0.668) and frequency (R = 0.508-0.817) domains, but the best correlation was for the high-frequency spectra (HF; R = 0.817). Our findings suggest that the short-term HRV remains stable and may be applicable for screening the variation in the heart rate of workers, although not all of the correlations were sufficiently strong.