Plant-based diets are increasingly adopted for health and environmental reasons, but they are associated with lower bone mineral density, higher fracture risk, and elevated bone turnover markers, particularly in women. These effects are partly explained by lower intakes of calcium and zinc, and higher concentrations of phytates and oxalates, substances that inhibit mineral absorption from plant foods. Evidence-based dietary strategies to support bone health in vegan populations beyond supplementation remain limited.

Fermented plant-based foods may help address this gap through two complementary mechanisms: first, by delivering live microorganisms that beneficially modulate gut microbiota and promote the production of short-chain fatty acids, which support mineral absorption and reduce bone resorption, and second, by reducing antinutritional factors such as phytates during microbial fermentation, thereby improving mineral bioavailability.

This study investigates whether the daily consumption of fermented plant-based foods, specifically lacto-fermented vegetables, calcium-fortified plant-based yogurt alternatives with live cultures, and Rhizopus-fermented tempeh, reduces bone resorption and improves calcium metabolism in premenopausal women following a vegan diet. Participants will follow each dietary condition (fermented or matched non-fermented control foods) for 12 weeks in randomized order, separated by an 8-week washout period. Blood, urine, and stool samples are collected at each study visit to assess bone turnover markers, gut microbiota composition, short-chain fatty acid production, inflammatory markers, and a range of metabolic and nutritional parameters.

Vegan diets are associated with lower bone mineral density and elevated fracture risk, partly due to reduced intake of calcium and zinc and higher concentrations of phytates and oxalates, which inhibit intestinal mineral absorption. Emerging evidence suggests that the gut microbiota plays a key regulatory role in bone metabolism through the gut-bone axis, primarily via production of short-chain fatty acids (SCFAs), which enhance mineral absorption, modulate immune responses, and suppress osteoclastogenesis. Fermented plant-based foods may beneficially modulate this axis by delivering live microorganisms and by reducing antinutritional factors through microbial phytase activity, thereby improving mineral bioavailability.

FERMBONE is a randomized, controlled, open-label, two-period crossover trial conducted at two sites: ETH Zürich (Switzerland) and Královské Vinohrady University Hospital (Prague, Czech Republic). Fifty premenopausal vegan women are enrolled (25 per site).

Study Design and phases:

After a screening visit, participants attend a baseline visit (week 0) to assess baseline characteristics and enter a 4-week run-in period during which fermented plant-based foods are excluded from the diet to standardize gut microbiota baseline. Participants are then randomized to one of two sequences: fermented foods in Phase I (weeks 4-16) followed by control foods in Phase II (weeks 24-36), or the reverse. The two 12-week intervention phases are separated by an 8-week washout period (weeks 16-24) during which the same dietary restrictions as in the run-in apply.

Intervention:

The fermented food intervention comprises three categories consumed daily: calcium-fortified fermented plant-based yogurt alternatives with live cultures, lacto-fermented vegetables including sauerkraut and kimchi, and Rhizopus-fermented tempeh, for a minimum combined total of 14 portions per week across at least two categories daily. The matched control condition consists of non-fermented plant-based equivalents: calcium-fortified plant-based milk, fresh raw vegetables, and cooked non-fermented legumes, matched at the group level for portion size, energy, protein, and calcium content. All study foods are commercially available and provided to participants.

Assessments:

Study visits are conducted at baseline and at the beginning and end of each intervention phase (weeks 0, 4, 16, 24, 36). At each visit, fasting venous blood samples, morning spot urine, and stool samples are obtained. Anthropometric measurements, blood pressure, standardized questionnaires, and a weighed 3-day dietary record are completed at each visit. A single DXA scan is performed during the run-in period to assess the baseline bone density.

Biological analyses:

Bone turnover markers (CTX, P1NP, ALP) and endocrine regulators (PTH, 25(OH)D) are measured from fasting serum. Further, urinary calcium, phosphate, and creatinine are measured. Gut microbiota composition is assessed by whole-genome metagenomic sequencing and fecal SCFAs are quantified by targeted metabolomics. Inflammatory and intestinal integrity markers (CRP, AGP, IL-6, iFABP), fecal pH and calprotectin, hemoglobin, and DNA methylation-based epigenetic aging markers are analyzed along with iron status markers (ferritin, soluble transferrin receptor) and metabolic parameters (glucose, insulin, lipid profile).

Inclusion Criteria:

• Female, aged 28-43years.

• Body mass index (BMI) between 18.5 and 24.9 kg/m².

• Premenopausal, with regular menstrual cycles. *

• Adherence to a vegan diet for ≥1 year, confirmed by questionnaire.

• Willingness to consume both fermented and non-fermented plant-based study foods.

• Ability to comply with all study procedures, including run-in, washout, sample collection, and clinic visits.

• Signed written and oral informed consent.

  • Regular menstrual cycles are defined as spontaneous menstrual bleeding occurring every 21-35 days, with cycle-to-cycle variability not exceeding 7-9 days over the prior three months, and bleeding duration of 3-8 days, in the absence of hormonal contraception or endocrine disorders.

Exclusion Criteria:

• Chronic diseases or conditions affecting bone metabolism (e.g., osteoporosis, thyroid disorders, chronic kidney and liver disease, menstrual cycles disorders affecting bone health, conditions associated with malabsorption).
• History of diagnosed eating disorders.
• Chronic inflammatory or gastrointestinal diseases (e.g., IBD, celiac disease).
• Recent antibiotic use (within 3 months prior to screening).
• Use of Vitamin D supplements exceeding 2000 IU/day.
• Abnormal DXA results (T-score ≤ -2.5).
• Pregnancy, intention to become pregnant, or lactation during the course of the study
• Use of hormonal contraceptives.
• Use of medications influencing bone metabolism (e.g., glucocorticoids, antiresorptives).
• BMI <18.5 or >24.9 kg/m².
• Known or suspected non-compliance, drug or alcohol abuse
• Inability to follow the procedures of the studies, e.g. due to language problems, psychological disorders, dementia, etc. of the participant or anticipated prolonged absence (e.g. extended travel).
• Participation in another clinical study within the last 3 months.
• Known allergy or intolerance to study foods.

Only female participants are eligible. The restriction to female participants is scientifically justified by the sex-specific effects of oestrogen on bone metabolism and the particular skeletal vulnerability of premenopausal vegan women. The biological mechanisms under investigation, including estrogen-dependent regulation of bone resorption, gut microbiota composition, and calcium metabolism, are sex-specific.

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