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Betaine supplementation has been shown to increase total testosterone concentrations in physically active men. However, its effects in women have not yet been investigated. The aim of the study was to evaluate the effect of eight weeks of betaine supplementation on sex hormone concentrations in women with overweight and obesity.

Methods: This was a double-blind, randomized controlled trial. Premenopausal women (18-45 years; n = 36) with overweight or obesity received either 3 g/day of betaine (BET) or a placebo (PLA) for eight weeks. Each participant attended two study meetings (before and after the supplementation) at the Department of Human Nutrition and Dietetics, Poznań University of Life Sciences, Poland between September 2024 and April 2025. Biological samples were collected before and after supplementation to assess sex hormone concentrations, which were measured using ELISA.

Results: There were no significant time × treatment interactions in the concentrations of sex hormones: plasma testosterone concentration: p = 0.496, salivary testosterone concentration: p = 0.882, plasma DHEA-S concentration: p = 0.498, plasma SHBG concentration: p = 0.775, plasma estradiol concentration: p = 0.378, plasma cortisol: p = 0.504.

Conclusions: No statistically significant effect of betaine supplementation was detected on sex hormone concentrations in premenopausal women with overweight or obesity, however larger and longer-term studies are warranted to confirm these findings. Clinical trial registry entry: https://clinicaltrials.gov/study/NCT06344377.