Statistical Analysis
All statistical analyses were performed with R software (version 4.1.2, R Core Team,2021). To investigate tDCS-induced changes in HRV, we calculated the change from baseline to the tDCS periods (5-min tDCS, during 0-back, and last 5-min tDCS) using delta scores (i.e: Δ score = RMSSD of 5-min tDCS minus RMSSD of baseline or HF-HRV of 5-min tDCS minus HF-HRV of baseline, etc). Outliers were inspected via boxplot distribution and three observations were excluded for the RMSSD data, while none was excluded for the HF-HRV (Sup. Material - Appendix 2). A linear mixed model (LMM) (‘lme4’ package) was used to assess the effects of tDCS on the HRV. The model included either delta RMSSD or HF-HRV scores as the dependent variable, with a fixed effect of tDCS protocol, time and their interaction (tDCS protocol*time). ‘Subject’ was employed as a random intercept (full statistical model: ‘RMSSD/HF-HRV ~ tDCS protocol*time + (1|Subject))‘. Pairwise analyses were performed using the ‘emmeans’ function.
Secondly, we investigated whether the HRV changes were associated with E-field magnitude induced by tDCS in the brain regions of interest. In the first step we employed LMM models having the RMSSD/HF-HRV delta scores as dependent variables, whereas the mean E-field in each region of interest were the fixed factors. ‘Subject’ was considered a random intercept. To investigate whether the mean individual E-fields were associated with the outcome, the same model was used but the variable ‘protocol’ was included as a fixed factor (RMSSD/HF-HRV ~E-field + tDCS protocol + (1|Subject)). In total, eight models were performed per analysis (4 brain regions of interest x 2 hemispheres). Therefore, multiple comparison corrections were conducted using the false discovery rate (‘stats’ package). Only the corrected p-values are presented here.
Exploratory analyses investigated whether the HRV changes could be influenced by baseline mood or state anxiety. For this analysis a LMM was fitted with the RMSSD/HF-HRV as the dependent variable and the interaction between mood and tDCS protocol (i.e: VAS baseline*tDCS protocol and STAI*tDCS protocol) as the independent variable (full model: RMSSD/HF-HRV ~ mood baseline*tDCS protocol + (1|Subject)). For all statistical tests, the significance level was set to alpha = 0.05.
Results
A total of 40 healthy volunteers were included. Two dropped-out after the first session and one was excluded due to non-normal alterations in the heart rate as indicated by a cardiologist. The final analyses were performed with 37 subjects (mean age = 24.3 years, standard deviation (SD) = 4.8), representing a total of 111 sessions being performed. Due to electrocardiogram artifacts during data collection (especially during the 0-back) a few time points were dropped from our dataset. Therefore, our analyses were conducted with a total of 103 time-points each for sham and 1.5mA, and 96 time-points for the 3.0mA protocol. For more information, a comprehensive table can be found in the Sup. Material - Appendix 3.