Normal range of 24 h heart rate variability (HRV) and its application in healthy pilots of different age and sex were investigated. 24 h HRV data in 118 healthy male and female pilots and ground crew were measured. Comparing with male pilots, SDNN, HRVi, RMSSD, HF and ATP values in females are obviously higher (P < 0.05). For HRV comparison analysis, the pilots were divided into three age groups. The results show that SDNN value of male pilots in group A is obviously higher than those in group B and C (P < 0.01). In female pilot group, the RMSSD, HF and ATP values of group C, are obviously higher than those of A and B (P < 0.01). It demonstrates that many HRV parameters show significant difference between male and female pilots, the HRV parameters in female pilot are obviously higher than those in males, especially for such parameters which reflect vagal nerve activity. Age factor effect is apparent in pilot\'s HRV data. Some HRV parameters in different age groups show significant difference. Most 24 h HRV parameters in pilots flying different types of aircraft show no significant difference.