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Background Physiotherapists often use thoracic spine mobilization (TSM) to reduce pain in patients with back disorders via a reduction of sympathetic activity. There is a “trade-off” in the activity of the sympathetic and parasympathetic nervous system activity. A sympathetic/parasympathetic balance (SPB) is needed to guarantee body homeostasis. However, body homeostasis is seldom considered as an aim of the treatment from the perspective of most physiotherapists. Strong empirical evidence for the effects of TSM on the SPB is still lacking. Some studies showed that spinal manipulation may yield beneficial effects on SPB. Therefore, it could be hypothesized that TSM is feasible and could influence SPB reactions. The primary aim was to describe the participants’ adherence to the intervention and to the measurement protocol, to identify unexpected adverse events (UAE) after TSM, to evaluate the best method to measure SPB parameters (heart rate variability (HRV), blood pressure (BP), heart rate (HR), skin perfusion and erythema) and to estimate the investigation procedure. The secondary aim was to assess the effects of TSM on SPB parameters in a small sample of healthy participants. Methods This crossover pilot study investigated TSM using posterior-anterior mobilization (PAM) and anterior-posterior mobilization (APM) on segments T6 to T12 in twelve healthy participants during two consecutive days. To evaluate feasibility, the following outcomes were assessed: adherence, UAE, data collection and data analysis. To evaluate the effect of TSM on SPB, HRV, BP, HR, skin perfusion and erythema were measured. Results The adherence was 100%. No UAE were reported. PAM showed larger effect sizes compared to APM in many secondary variables. Conclusions Although 100% maximal adherence was reached and no UAE were observed, data recording in future studies should be done during a second time interval while the data transfer from device to the computer software should occur immediately after completion of each participant’s measurement. The results of this pilot study suggest that PAM can reduce HRV HF and HRV ratio LF/HF and increase HR. Trial registration ClinicalTrail.gov (NCT02832141).