*4.2. Study 2 Design*

This double-blind placebo-controlled trial was prospectively registered with the Australian New Zealand Clinical Trial Registry (ACTRN12617001010381) and approved by the University of Wollongong Human Research Ethics Committee (approval 2017/101). Participants were enrolled in the study if they were 18 years or over, had an overweight BMI (25−<30 kg/m2), had not recently consumed antibiotics (previous two months), and were not pregnant.

Participants with an inflammatory skin condition were also included in the study, although this subgroup was analyzed separately (data presented in Roach et al., anticipated publication 2022).

Participants were advised to maintain their usual diet and medication regimes. The sample size was determined using a power calculation with data from Study 1 and an online calculator (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html, accessed on 7 June 2017). The change in non-HDL cholesterol that was observed in Study 1 (−0.37 mmol/L) was used as the primary outcome, the test was two-sided, with a power of 80% and alpha set at 0.05. A sample of ten per group was calculated as being statistically sufficient, with a view to recruit up to 30 per group to allow for participants that may potentially withdraw consent and missing data. Furthermore, 17 per group and 19 per group were required to detect changes in LDL-cholesterol and total cholesterol, respectively. Overweight participants were recruited as they showed the greatest response to the treatment in Study 1. A 2 g dose of SXRG84 was selected, as this dose reduced non-HDL cholesterol in Study 1.

Participants were randomized into two regimes: placebo group for six weeks and then crossed to the treatment group for six weeks (AB) or vice versa (BA) (Figure 7). Therefore, all participants consumed both the treatment and the placebo during the trial, but they were blinded as to when they consumed each treatment. Allocation of the treatment regime and labeling of the study treatments were completed by an individual independent of the

study. There was no washout between the first six weeks and then second six weeks of the trial.

**Figure 7.** Study Design Regime AB, placebo followed by SXRG84 treatment. Regime BA, SXRG84 treatment followed by placebo.
