The research behind Protocol One, ingredient by ingredient
What each study found, who was studied, who paid for it, and what it doesn't show, including the places where the evidence is thinner than supplement marketing usually admits.
What research is Protocol One based on?
Protocol One's ingredient choices rest on a few small studies of individual ingredients; no study has tested Protocol One itself. The most direct evidence compares iron forms in 78 pregnant women and zinc forms in 12 women [1, 2], and the probiotic trial was written by researchers from the company that makes the ingredient [3]. Each entry below says what a study found and what it doesn't show.
Key numbers
- Iron: in a trial of 78 pregnant women, 25 mg of ferrous bisglycinate caused significantly fewer digestive complaints than 50 mg of ferrous sulfate [1].
- Zinc: in a single-dose study of 12 women, zinc bisglycinate was 43.4% more bioavailable than zinc gluconate [2].
- Folate: the CDC says people with MTHFR gene variants can process folic acid, so methylfolate is a formulation choice, not a necessity [4].
- Probiotic: in a 4-week trial of 70 adults with gas and bloating, by researchers from the ingredient's maker, LactoSpore improved symptom scores more than placebo [3].
What research is behind Protocol One's iron form?
Protocol One uses ferrous bisglycinate, which caused significantly fewer digestive complaints than ferrous sulfate in a trial of 78 pregnant women [1]. A 2023 review of 17 trials points the same way for pregnant women but rates the evidence very low certainty [5]. Our full comparison of iron bisglycinate and ferrous sulfate covers doses, reliability and how much iron adults need.
- In Protocol One
- 18 mg of iron as ferrous bisglycinate chelate [6].
- What the study found
- Two randomized, double-blind trials in pregnant women were reported together (482 women in total). The bisglycinate comparison came from one of them: 78 women took 25 mg of iron as ferrous bisglycinate or 50 mg as ferrous sulfate daily until delivery. Bisglycinate had significantly fewer digestive complaints. Ferrous fumarate at 40 mg, tested in the other trial, had a complaint profile similar to sulfate [1].
- Black stools
- Reported by 8% on bisglycinate, 22% on ferrous fumarate and 31% on ferrous sulfate. Black stools rose with iron dose, and the authors did not count them as a side effect [1].
- Who was studied
- Healthy pregnant women in Denmark, starting in mid-pregnancy [1].
- The meta-analysis
- 43 randomized trials with 6,831 adults compared ferrous sulfate with placebo or intravenous iron. Against placebo the odds ratio for digestive side effects was 2.32, and the authors found no clear link to dose [7].
- What it doesn't show
- The trial doses weren't equal, no one was taking a GLP-1 medication, and the meta-analysis did not test bisglycinate at all.
Multivitamins use a range of iron forms. Centrum Women, for example, lists ferrous fumarate [8].
Is zinc bisglycinate better absorbed than zinc gluconate?
Zinc bisglycinate was better absorbed than zinc gluconate in one small study. Twelve healthy women each took a single 15 mg dose of both forms a week apart, and bisglycinate's bioavailability, measured from blood zinc, was 43.4% higher [2]. That is a one-dose blood measurement, not evidence of better zinc status over months.
- In Protocol One
- 20 mg of zinc as zinc bisglycinate chelate [6].
- What the study found
- In a randomized crossover design, oral bioavailability of zinc was 43.4% higher with bisglycinate than with gluconate, and bisglycinate was reported as safe and well tolerated [2].
- Who was studied
- 12 healthy adult women in France [2].
- Who paid
- Co-authors included researchers from Laboratoire Oenobiol, a supplement company [2].
- What it doesn't show
- Long-term zinc status, comparisons with other zinc forms, or effects in people taking GLP-1 medications.
Do you need methylfolate instead of folic acid?
The CDC says no. People with MTHFR gene variants can process all types of folate, including folic acid, and people with two copies of the common C677T variant average only about 16% lower blood folate on the same intake [4]. The CDC adds that 400 mcg of folic acid a day raises blood folate regardless of genotype, so Protocol One's L-5-MTHF is a formulation choice, not a fix for folic acid.
- In Protocol One
- 400 mcg DFE of folate as L-5-MTHF calcium salt [6].
- What the CDC says
- The advice that people with an MTHFR variant should avoid folic acid and take another form is not true, and folic acid is the only type of folate shown to help prevent neural tube defects [4]. The CDC's guidance for clinicians adds that there are no clinical recommendations to test for MTHFR or to change folate intake based on the result [9].
- What it means for you
- If you could become pregnant, talk with your prescriber about folic acid. Protocol One is not intended for use during pregnancy [6].
Is methylcobalamin better than cyanocobalamin?
The evidence for methylcobalamin is thinner than supplement marketing suggests. A 2025 review reports animal studies in which cyanocobalamin was excreted in urine at about three times the rate of methylcobalamin, with 13% more B12 stored in the liver on the methyl form [10]. Those are animal results, and they don't show that methylcobalamin works better in people.
- In Protocol One
- 525 mcg of vitamin B12 as methylcobalamin [6].
- What the review says
- The retention findings come from animal studies. The review's authors nonetheless recommend prioritizing methylcobalamin [10].
- What it doesn't show
- Better absorption, better B12 status, or better symptoms in people. We haven't found human data we can verify on those questions.
Does LactoSpore help with gas and bloating?
LactoSpore improved gas and bloating scores more than placebo in one trial by researchers from its manufacturer. Seventy adults with functional gas and bloating took 2 billion spores a day of Bacillus coagulans MTCC 5856 or placebo for 4 weeks [3]. The placebo group improved too, no participants were taking GLP-1 medications, and Protocol One lists 6 billion CFU rather than the 2 billion studied [6].
- In Protocol One
- LactoSpore (Bacillus coagulans MTCC 5856), 166 mg, listed as 6 billion CFU [6].
- What the study found
- Digestive symptom scores and patients' global ratings improved significantly more with the probiotic than with placebo, and no adverse events were reported. Of 70 people enrolled, 66 finished [3].
- Who was studied
- Adults with functional gas and bloating, at hospitals in southern India [3].
- Who paid
- All five authors are affiliated with Sami-Sabinsa Group or Sabinsa Corporation, which makes LactoSpore and supplied it for the trial [3].
- What it doesn't show
- Effects beyond the 4-week study period [3], effects in GLP-1 users, or that a higher spore count works better.
What about the ginger, peppermint and enzyme blend?
Protocol One contains a 325 mg Digestive Comfort & Soothing Blend of ginger root extract, peppermint leaf extract, bromelain and DigeZyme [6]. We don't cite ginger or peppermint trials for this blend, and we don't claim it relieves nausea, bloating or any other specific symptom.
Other ingredients, and why we make no claim for them
- BioPerine (black pepper extract, 5 mg): its supplier reports absorption studies with other nutrients [11], but we haven't found peer-reviewed human data for the nutrients and doses in Protocol One, so we make no claim for it [6].
- Magnesium (200 mg as magnesium oxide and magnesium citrate): included for general intake, with no claim about the form [6].
- Vitamin D3 (50 mcg) and vitamin B6 (26 mg as P5P): included as general nutrients [6]. For vitamin D on a GLP-1, see our guide to nutrient deficiencies on GLP-1 medications.
- Ashwagandha root extract (part of a 126 mg complex): a traditional herb with no claim made here. Don't use it if pregnant or nursing, and check with your provider if you have a thyroid or liver condition [6].
What no study has tested
- No study has tested Protocol One itself, in GLP-1 users or anyone else.
- None of the ingredient studies on this page enrolled people taking GLP-1 medications.
- Several were small, single-dose, animal-based or run by an ingredient maker, as each entry notes.
- When we read a study that changes one of these conclusions, we update this page and its review date.
About this page
Understory Nutrition wrote this page and sells Protocol One, a daily supplement designed to be taken alongside a GLP-1 medication. Every entry links to the original study so you can read it yourself.
Sources
- Milman NT, Bergholt T. Low-dose prophylactic oral iron supplementation (ferrous fumarate, ferrous bisglycinate, and ferrous sulphate) in pregnancy is not associated with clinically significant gastrointestinal complaints: results from two randomized studies. Journal of Pregnancy. 2024;2024:1716798. doi:10.1155/2024/1716798 Full text. Two randomized, double-blind trials in healthy pregnant women (404 and 78 women).
- Gandia P, Bour D, Maurette JM, et al. A bioavailability study comparing two oral formulations containing zinc (Zn bis-glycinate vs. Zn gluconate) after a single administration to twelve healthy female volunteers. International Journal for Vitamin and Nutrition Research. 2007;77(4):243-248. doi:10.1024/0300-9831.77.4.243 PubMed. Randomized crossover, 12 women, single dose. Co-authors from Laboratoire Oenobiol.
- Majeed M, Nagabhushanam K, Paulose S, Arumugam S, Mundkur L. The effects of Bacillus coagulans MTCC 5856 on functional gas and bloating in adults: a randomized, double-blind, placebo-controlled study. Medicine. 2023;102(9):e33109. doi:10.1097/MD.0000000000033109 Full text. 70 adults, 4 weeks, southern India. All authors affiliated with the Sabinsa group, which makes LactoSpore.
- Centers for Disease Control and Prevention. MTHFR Gene Variant and Folic Acid Facts. cdc.gov. Accessed September 16, 2026.
- Fischer JAJ, Cherian AM, Bone JN, Karakochuk CD. The effects of oral ferrous bisglycinate supplementation on hemoglobin and ferritin concentrations in adults and children: a systematic review and meta-analysis of randomized controlled trials. Nutrition Reviews. 2023;81(8):904-920. doi:10.1093/nutrit/nuac106 PubMed. 17 randomized trials, 2,191 people. Funded by the University of British Columbia; no relevant interests declared.
- Understory Nutrition. Daily Micronutrient & Digestive Protocol, Supplement Facts label. Product page.
- Tolkien Z, Stecher L, Mander AP, Pereira DIA, Powell JJ. Ferrous sulfate supplementation causes significant gastrointestinal side-effects in adults: a systematic review and meta-analysis. PLoS One. 2015;10(2):e0117383. doi:10.1371/journal.pone.0117383 Full text. 43 randomized trials, 6,831 adults; comparators were placebo or intravenous iron.
- Pfizer. Centrum Women Multivitamin/Multimineral Supplement, product labeling. labeling.pfizer.com. Manufacturer label; ingredient list checked September 16, 2026.
- Centers for Disease Control and Prevention. Folic Acid: Facts for Clinicians. cdc.gov. Accessed September 16, 2026.
- Vitamin B12: a comprehensive review of natural vs synthetic forms of consumption and supplementation. Cureus. 2025. Full text. Narrative review; the retention figures it reports come from animal studies.
- Sabinsa. BioPerine: Vitamin B6 (supplier website). bioperine.com. Supplier-published summary, not a peer-reviewed journal article.
These statements have not been evaluated by the Food and Drug Administration. Understory Nutrition products are not intended to diagnose, treat, cure, or prevent any disease. This page is general information, not medical advice. Talk with your prescriber before starting a supplement, especially alongside a prescription medication.