This study examined the effect of 27 hours of sleep deprivation on heart rate variability (HRV) according to the Korean military fitness test. The Korean military fitness test has the following five grades: excellent, 1st grade, 2nd grade, 3rd grade, and 4th grade. A high fitness level group included individuals with an excellent grade, and a low fitness level group included individuals with a grade lower than excellent grade. Thirty-eight military officers participated in this study, and they underwent 27 hours of sleep deprivation. Because of sleep deprivation, there was a significant difference in very low frequency (VLF)(p=0.014) between the high and low fitness level groups. Additionally, there were interactions between VLF(p=0.003), low frequency(p=0.024), high frequency(p=0.020), total HRV(p=0.030), and standard deviation of all normal R-R intervals(p=0.005). In conclusion, cardiorespiratory fitness, which is strongly related with heart rate variability, seems to be an important factor to reduce cardiac stress caused by sleep deprivation.