Quantification of physical fatigue remains a challenge. We hypothesized that its effects on central autonomic nervous system activity could be explored for such a quantification. To test this relationship, we prospectively measured central autonomic nervous activity through nocturnal heart rate variability (HRV) in six French garbage collectors, aged 32.1+/-4.3 years, twice a week during 3 consecutive weeks of work, and during the following week of rest. Eight healthy sedentary males formed a control group. HRV indices were calculated by applying standard temporal domain and wavelet transform analyses to standard ECG recordings. During the 3 consecutive weeks of work, there was a significantly progressive decrease in HRV indices, particularly pNN50 (-34.2%, P<0.05), as well as the high (-33.3%, P<0.05) and low (-22.2%, P<0.01) frequency components of wavelet transform, while there was an increase, although non-significant, of the ratio of low to high frequencies (9.1%). During the resting period, there was a significant recovery of HRV indices, notably of its high (50.0%, P<0.05) and low (28.6%, P<0.05) frequency components. No such changes occurred in the control group. A central signature of cumulated physical fatigue can thus be detected and quantified through nocturnal autonomic nervous system activity. Its characteristics are those of a progressive parasympathetic withdrawal.