## 2 Materials and Methods
### Experimental Procedures  
Participants received milk thistle seeds (HerbaNordPol-Gdarask LLC, Nowy Staw, Poland) as a source of silymarin. Taking this into account, the daily dose of milk thistle was calculated based on the required silymarin dose. In clinical studies, various silymarin doses are applied, differing from 210 mg to 1200 mg a day [33]. At the same time, silymarin is indicated to be well tolerated, without any side effects, when applied in doses from 200 mg to 900 mg, but doses higher than 1500 mg may cause excessive bile production and laxative effects [34]. The silymarin content in the applied grinded milk thistle seeds was assessed based on the method of high-performance liquid chromatography (HPLC) [25] in a certified laboratory of the Institute of Natural Fibres and Medicinal Plants in Poznan (Poland), and it was observed to amount to 2.96%, including silybins A and B (1.76%), silychristins (0.78%), isosilybins A and B (0.40%) and silydianin (0.06%). On the basis of this assessment, participants received a daily dose of 7.1 g of milk thistle (210 mg of silymarin). They received it for 3 months, and they were provided daily servings in air-tight containers. They received them during their medical and dietary checkup, every 2 weeks for the following 14 days.  
Participants received commercially available grinded milk thistle seeds and were instructed to consume them within their regular dishes, as the product is characterized by a neutral taste, smell and flavor; thus, it may be added to any product. They were informed that it may be added, e.g., to yoghurt, soup, salad, etc.  
At the same time, participants received dietary and physical activity recommendations as essential elements of the therapy for NAFLD and obesity [16; 17]. Their diet, physical activity and milk thistle consumption were monitored every 2 weeks, and not following the dietary and physical activity recommendations they were encouraged to follow was not a reason to exclude participants from the study.  
The dietary and physical activity recommendations were individually developed for each participant based on their dietary and physical activity baseline assessments, as well as their energy requirement that was calculated based on the Harris-Benedict equation [35]. They were recommended to follow a diet with a 600 kcal energy deficit per day, which is in agreement with recommendations for NAFLD patients to meet the energy deficit of 500 kcal-750 kcal within their diet [36]. Based on the 3-day dietary record, provided by each participant, the usual intake was established and individual dietary recommendations were planned. The recommendations included the energy deficit, an adequate number of meals per day (4-5), intervals between meals of about 3 h, changes of food product choice (increased intake of whole grain cereals, vegetables, no sweets and fast foods) and recommended methods of thermal treatment (cooking or baking instead of frying), which is in agreement with general recommendations for NAFLD patients and within the Mediterranean dietary pattern [37]. The diet that was planned for each participant was individualized, taking into account concurrent diseases. Each patient received individual recommendations as well as individual menus with information on how to use them and how to diversify them.  
At the same time, physical activity recommendations were developed to obtain an additional deficit of 400 kcal, which is also in agreement with recommendations for NAFLD patients [36]. The physical activity recommendations were associated with moderate intensity physical activity as recommended for NAFLD patients [38], and the recommended activity was based on a target daily number of steps, as well as encouraging patients to walk, jog, cycle and swim, if possible [36]. Patients were recommended to engage in physical activity for at least 30-60 min and at least 3-5 days a week, and the detailed recommendations were based on guidelines for NAFLD patients [39].