2.7. Statistical Analysis
depression, anxiety
Statistical analyses were conducted on 45 subjects that were randomized to one of the two arms (Psychological endpoints related to acute stress (PANAS, STAI-6, and VAS) and general assessments (PSS-14, HADS, GSRS, and PSQI) were used to assess the effect of the intervention by estimating the treatment difference (probiotic vs. placebo) at the post-intervention visit by an analysis of covariance (ANCOVA) model including treatment as independent variable as well as the three covariates: gender, DASS-42, and the pre-treatment value considered as baseline.Since saliva cortisol measurements feature multiple timepoints, data were summarized using four parameters that were then analyzed using the same ANCOVA model: area under the curve (AUCg), area under the curve incremental to the first timepoint considered as baseline (AUCi), maximum concentration (Cmax), and incremental maximum concentration (ICmax). For the autonomic parameters, the variables of interest were the changes from the pre-stressor value that was considered as baseline. These differences vs. baseline were analyzed using a linear mixed model with treatment, visit, treatment*visit, gender, DASS-42, and baseline (pre-stressor timepoint) as fixed and subject as random effects.For endpoints not meeting the model assumptions of normality, a Box-Cox transformation was applied. In such cases, a back-transformation with bias adjustment was applied to obtain an estimate of the effect on the original scale. When models did not converge or model assumptions were severely violated, a Wilcoxon rank-sum test was used together with the Hodges–Lehmann estimator and an associated nonparametric 95% confidence interval.In order to summarize the main findings of the whole study in a single descriptive figure, a principal component analysis (PCA) was performed on the difference between baseline and post-intervention of heart rate and PSS-14 as active variables and PSQI total score, PSQI subjective sleep quality score, HADS depression score, HADS anxiety score, cortisol acute response to experimental stress, cortisol awakening response, RMSSD at baseline (first period), RMSSD After 8 period, and DASS-42 baseline as passive variables. This PCA was performed on z-score-transformed variables.The statistical significance level was fixed at 5%, without adjustments for multiplicity, due the exploratory nature of the study. All analyses were performed using SAS BASE 9.4/SAS STAT 15.1 on the SAS Life Science Analytics Framework (SAS LSAF, SAS Institute Inc., Cary, NC, USA) version 5.2.3.