Regular and long-term shift work is associated with a number of chronic diseases. There is some evidence that shift work also has an influence on the autonomous nervous system. Studies that have examined the effect of shift work on heart rate variability (HRV) have not taken into account age and gender. Therefore, the aim of this study was to investigate the influence of night shift based on 24 h long-term analyses carried out on non-night shift days with a matched control group for age and gender. In total, 172 (128 male, 44 female) healthy shift night workers were compared with subjects from a non-night shift worker group at a ratio of 1:1, forming matched pairs based on the subjects' sex and age. HRV parameters were analyzed based on 24 h ECG recording. An analysis of the HRV parameters showed only a small difference but without statistical significance between the two groups with regard to all of the HRV parameters examined (SDNN, RMSSD, NN50, pNN50, VLF, LF, HF, LF/HF, DFA1, and DFA2). An analysis of the subgroup of subjects who had only worked night shifts for a minimum of 10 or 20 years, with the respective pairs matched by age and gender, did not reveal any significant differences between the HRV parameters of employees working night shifts and those not working night shifts. Taking into account qualitative aspects of HRV analysis, this study was thus able to show that working night shifts for many years may not have as big an influence on HRV as had been assumed so far.