The onset of fatigue disrupts the functioning of the autonomic nervous system (ANS), potentially elevating the risk of life-threatening incidents and impairing daily performance. Previous studies mainly focused on physical fatigue (PF) and mental fatigue (MF) effects on the ANS, with limited knowledge concerning the influence of physical-mental fatigue (PMF) on ANS functionality. This study aimed to assess the immediate impact of PMF on ANS function and to compare its effects with those of PF and MF on ANS function. Thirty-six physically active college students (17 females) without burnout performed 60-min cycling exercises, AX-Continuous Performance Task (AX-CPT), and cycling combined with AX-CPT to induce PF, MF, and PMF respectively. Subjective fatigue levels were measured using the Rating of Perceived Exertion scale and the Visual Analog Scale-Fatigue. Heart rate variability was measured before and after each protocol to assess cardiac autonomic function. The proposed tasks successfully induced PF, MF, and PMF, demonstrated by significant changes in subjective fatigue levels. Compared with baseline, PMF decreased the root mean square of successive differences (RMSSD) between normal heartbeats (P < 0.001, d = 0.50), the standard deviation of normal-to-normal RR intervals (SDNN) (P < 0.01, d = 0.33), and the normalized high-frequency (nHF) power (P < 0.001, d = 0.32) while increased the normalized low-frequency (nLF) power (P < 0.001, d = 0.35) and the nLF/nHF ratio (P < 0.001, d = 0.40). Compared with MF, PMF significantly decreased RMSSD (P < 0.001, η2 = 0.431), SDNN (P < 0.001, η2 = 0.327), nLF (P < 0.01, η2 = 0.201), and nHF (P < 0.001, η2 = 0.377) but not the nLF/nHF ratio. There were no significant differences in ∆HRV (i.e., ∆RMSSD, ∆SDNN, ∆nLF/nHF, ∆nLF, and ∆nHF), heart rate, and training impulse between PF- and PMF-inducing protocols. Cognitive performance (i.e., accuracy) in AX-CPT during the PMF-inducing protocol was significantly lower than that during the MF-inducing protocol (P < 0.001, η2 = 0.101). PF and PMF increased sympathetic activity and decreased parasympathetic activity, while MF enhanced parasympathetic activity. |