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Dietary supplementation has the potential to selectively modify its composition. Nigella sativa seed oil (NSSO), which contains the bioactive compound thymoquinone (TQ), has antimicrobial and immunomodulatory properties, but human data on NSSO-associated gut microbiome changes and urinary TQ detectability after repeated oral exposure remain limited. In this prospective, single-center, randomized open-label pilot study, eight healthy adults were assigned to a treatment group (\n                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u20094) receiving TQ-containing NSSO capsules (two capsules, three times daily for 20 days) or a control group (\n                    <jats:italic>n<\/jats:italic>\n                    \u2009=\u20094) without supplementation. Fecal samples were collected at five time points and analyzed by 16\u00a0S rRNA gene sequencing. Alpha and beta diversity, exploratory differential abundance, and machine learning analyses were performed. Food diaries were reviewed descriptively. Urine samples obtained at two time points were analyzed using Gas Chromatography Mass Spectrometry (GC-MS) to detect TQ and its metabolites. No significant differences in alpha or beta diversity were observed between groups, proving a stable microbiome composition. However, specific taxa increased or decreased, among them\n                    <jats:italic>Mediterraneibacter lactaris<\/jats:italic>\n                    (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009=\u20099\u2009\u00d7\u200910\u207b\u2075) and\n                    <jats:italic>Terrisporobacter mayombei<\/jats:italic>\n                    (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009=\u20093\u2009\u00d7\u200910\u207b\u2074), respectively. TQ and\/or TQ-H\u2082 were qualitatively detected in urine samples from the treatment group after repeated NSSO intake. This pilot study suggests that repeated NSSO intake is feasible for longitudinal microbiome and urinary metabolite sampling and provides preliminary evidence for taxon-specific changes in healthy adults. Urinary detection of TQ\/TQ-H\u2082 confirms detectability following repeated NSSO intake but does not permit pharmacokinetic conclusions. These findings may guide the design of future blinded, placebo-controlled, diet-controlled perioperative microbiome studies.\n                  <\/jats:p>\n                  <jats:p>Trial Registration: The trial was retrospectively registered at ClinicalTrials.gov (NCT07450807) on 4 March 2026.<\/jats:p>","DOI":"10.1038\/s41598-026-69567-3","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T03:23:13Z","timestamp":1789442593000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A prospective randomized controlled pilot study of gut microbiome modulation by Nigella sativa seed oil and thymoquinone metabolism in healthy volunteers: implications for gastrointestinal surgery"],"prefix":"10.1038","volume":"16","author":[{"given":"Ahmad Firas","family":"El-Banna","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Howard","family":"Junca","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arndt","family":"Steube","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dirk K.","family":"Wissenbach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Utz","family":"Settmacher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claus","family":"Schneider","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aysun","family":"Tekba\u015f","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,9,15]]},"reference":[{"issue":"12","key":"69567_CR1","doi-asserted-by":"publisher","first-page":"2019","DOI":"10.1007\/s10482-020-01474-7","volume":"113","author":"EZ Gomaa","year":"2020","unstructured":"Gomaa, E. 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