Introduction: Physiotherapy treatment is a common intervention for low back pain (LBP) patients. These interventions have been related to physiological effects in the central nervous system. Thus, the aim of this study was to analyze the effect of physiotherapy treatment in patients with LBP in the autonomic nervous system activation and subjective pain perception of patients. Methods: A total of 30 male subjects diagnosed with non-specific subacute LBP received a 50 min session consisting of (a) a manual therapy based on joint mobilization and soft tissues techniques in the lumbo-pelvic area, (b) a stretching program, and (c) motor control exercises of the core muscles. The autonomic modification of participants was assessed prior to and after the physiotherapy treatment. Results: Heart rate variability (HRV) analysis reported a significant increase in average RR (p = 0.001), RMSSD (p = 0.008), LRMSSD (p = 0.001), SDNN (p = 0.005), and PNN50 (p = 0.024) after the session. Frequency-domain measures showed a significant increase in LF (p = 0.030) and HF (p = 0.014), and a decrease in LF/HF ratio (p = 0.046). A significant decrease was found in minimum HR values (p = 0.001) and average HR (p = 0.001). Moreover, maximal HR decreased its value from 116.7 ± 26.1 to 113.7 ± 40.8 after intervention. In addition, subjective pain perception (VAS scores) was significantly lower (p = 0.001) in the post-session assessment. Conclusions: Physiotherapy treatment produced an increase in parasympathetic nervous system activation and a decrease in subjective pain perception in non-specific subacute LBP patients.