Abstract
Background
Hypertension affects 1 in 3 Australian adults and is a major contributor to stroke and myocardial infarction. Despite the availability of various medications, over 50% of patients struggle to control their blood pressure (BP) due to nonadherence, difficulty finding the right treatment, or avoidance of medication. Diet plays a key role in cardiovascular health, with high-fiber intake shown to lower BP through the production of short-chain fatty acids (SCFAs), metabolites formed during fiber fermentation. A previous clinical trial found that incorporating a specific type of resistant starch – high amylose maize starch enriched with acetate and butyrate (HAMSA/B) — significantly reduced blood pressure in hypertensive patients. While the exact mechanism remains unclear, SCFAs may influence BP via the gut-brain axis by reducing muscle sympathetic nerve activity (MSNA), which has vasoconstrictor function and is elevated in hypertensive individuals.
Methods
The GRAINS-BP clinical trial will recruit 29 adults with untreated hypertension (≥ 140/90 mmHg) for a double-blind, cross-over study using HAMSA/B administered as a shake delivered twice daily. The assessment period will span 48 days and include four visits at the Alfred Hospital, Melbourne, Australia. At visit 1, baseline measurement will be taken, including muscle sympathetic nerve activity (MSNA) and electrocardiogram (ECG). Participants will be randomised to the order in which they receive the placebo and HAMSA/B shake. Each intervention period will last 14 days, separated by a 20-day washout before participants cross over to the alternate treatment. At each visit, faecal, urine, and blood samples will be collected to investigate microbiome, cardiometabolic, renal, and immune profiles, respectively. Univariate and multivariate statistical analyses will be conducted to examine treatment effects and to identify correlations among microbiome, cardiometabolic, renal, and immune outcomes.
Discussion
This project’s primary outcome is change in autonomic nervous system activity. Secondary outcomes include 24-h BP, gut microbiome composition, SCFA levels, immune cell profiles gastrointestinal transit time, quality of life, anxiety and depression symptoms, cognitive function, gut barrier integrity, and circulating micro- and nanoplastics. Findings from this study will reveal whether SCFAs reduce blood pressure by their influence on the autonomic nervous system.
Trial registration
This trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12625000557437).
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s13063-026-09911-0.
Keywords: Gut microbiome, Gut-brain axis, Sympathetic nerve activity, Hypertension, Short-chain fatty acids, Diet, Fibre, Resistant starches, Randomised controlled trial
Introduction
Background and rationale < 6a >
Hypertension, or high blood pressure (BP), is the foremost cause of death and is expected to remain so until 2050 [1]. Despite the wide range of antihypertensive medications available, approximately 50% of hypertensive individuals do not achieve adequate BP control [2]. Uncontrolled hypertension can lead to severe and possibly fatal conditions such as stroke, myocardial infarction, and chronic kidney disease, resulting in significant economic impacts [3]. Diet plays a vital role in regulating BP [4]. A high-fiber diet lowers BP via short-chain fatty acids (SCFAs) — mainly acetate, propionate and butyrate — metabolites produced by the fermentation of dietary fibre by the gut microbiome and absorbed into the circulation [4].
Our group found that a high-fiber diet or supplementation with the SCFA acetate in drinking water lowered BP and reduced related complications in the deoxycorticosterone (DOCA)-salt hypertension mouse model [5]. Furthermore, we and others have shown that butyrate lowers BP in angiotensin II-induced hypertensive mouse models [6–8], possibly by improving the gut epithelial barrier, leading to reduced passage of microbial components from the gut to the circulation that lead to inflammation [9]. Recently, the potential of SCFAs to reduce BP in humans was tested in a double-blind, placebo-controlled, randomized crossover trial [10]. A 3-week intervention with 40 g/day of fibre chemically enriched with acetate and butyrate, called high amylose maize starch that was acetylated and butyrylated (HAMSA/B), delivering high levels of SCFAs to the colon, reduced 24-h systolic BP by 6.1 mmHg compared to the placebo arm in twenty untreated hypertensive participants [10]. Similarly, daytime and nighttime systolic BP both decreased as assessed by 24-h ambulatory BP measurement [10]. This drop in BP was observed as little as 10 days after the intervention and was associated with a twofold increase in acetate and a 14-fold increase in butyrate [10].
Despite these promising findings, the precise mechanisms by which fibre and SCFA supplementation lower BP, particularly in humans, remain largely unknown. We propose a novel hypothesis that microbial metabolites influence BP by activating central mechanisms through pathways termed the ‘gut-brain axis’. SCFAs act as crucial intermediaries in the gut-brain axis and are integral to the neurobiological processes underlying mental illnesses such as depression and anxiety [11]. SCFAs can trigger the release of neurotransmitters by neuroendocrine cells that line the gastrointestinal tract, called enteroendocrine cells (EECs). These neurotransmitters can transmit signals to the brain via the vagus nerve and spinal afferents. The brain in turn interprets these signals and provides feedback to organs such as the spleen, modulating immune response.
Hypertensive animals and patients have greatly elevated sympathetic vasoconstrictor drive to the peripheral resistance vessels, such as those in the skeletal muscle vascular bed [12]. This can be measured directly in humans by inserting a tungsten microelectrode into an accessible peripheral nerve and recording spontaneous bursts of muscle sympathetic nerve activity (MSNA); each burst of MSNA leads to vasoconstriction, and the resultant reduction in blood flow and increase in BP can be quantified as sympathetic vascular transduction [13]. Conversely, until recently, there have been no direct measures of parasympathetic activity. Cardiac parasympathetic outflow has instead been inferred from indices of heart rate variability (HRV) derived from electrocardiographic recordings, such as high‑frequency HRV or the root mean square of successive differences (RMSSD), which quantifies short‑term beat‑to‑beat variability. These measures reflect the magnitude of fluctuations in cardiac vagal activity rather than the absolute level of ongoing parasympathetic tone, and thus provide only an indirect assessment of autonomic activity. Understanding these mechanisms is critical for developing new therapies. Understanding the influence of SCFAs on the gut-brain axis could pave the way for new treatments for hypertension and other conditions linked to this axis, such as irritable bowel syndrome (IBS) and neuropsychiatric disorders like depression and anxiety [14].
Objectives < 7 >
To determine whether 14-day supplementation with the HAMSA/B shake (a source of SCFA) lowers blood pressure in humans and to explore how this effect may be influenced by gut microbiome composition, immune cell activity, and gastrointestinal transit time.
To determine whether SCFAs improve quality of life, reduce symptoms of anxiety, depression, and improve cognition.
To determine if SCFAs improve gut barrier markers, thus reducing the passage of microbial substances and microplastics from the gut to the circulation.
Trial design < 8 >
GRAINS-BP is a double-blind randomized placebo-controlled crossover phase II clinical trial in people with untreated hypertension (according to Australian guidelines and definitions [15]), with a 1:1 allocation ratio and an exploratory framework (Fig. 1).
Fig. 1.
Schematic diagram of participants' visits. ABPM, ambulatory blood pressure monitoring; BP, blood pressure; HR, heart rate; SCFA, short-chain fatty acid. Created in part using biorender.com’
Methods: participants, interventions and outcomes
Study setting < 9 >
This is a single-center trial. The clinical trial facilities are in Monash-operated laboratories at The Alfred Centre, Melbourne, Australia.
Eligibility criteria < 10 >
Participants will be screened based on the inclusion and exclusion criteria below. These were designed based on our previous trial.
Inclusion criteria
18–75 years of age;
BMI 18.5–35 kg/m2;
Either sex
Office BP and/ or home BP categorised as hypertension (systolic BP ≥ 140 mm Hg and/or diastolic BP ≥ 90) according to the Australian Heart Foundation guidelines [16];
Intake of whole grains lower than 2 servings per day (females) and 3 servings per day (males)*.
*The threshold for whole grain intake was informed by findings from our team that participants who responded to the intervention in our previous trial [11] had lower whole grain intake [17].
Exclusion criteria
Use of anti-hypertensive medication (current or in the past 4 weeks);
24-h BP systolic BP > 160 and/or diastolic BP > 100 mmHg;
Recent use of oral antibiotics (< 3 months);
Recent use of probiotics (< 4 weeks);
Current use of laxatives;
History of cardiometabolic disorders (stroke, atrial fibrillation (AF), heart failure, and ischaemic heart disease) or prior diagnosis of cardiac arrhythmia, heart rhythm disorders;
Presence of type 1 or type 2 diabetes;
Pregnancy, breastfeeding or plans to conceive during the course of the trial;
Presence of gastrointestinal diseases or digestive conditions (including inflammatory bowel disease, lactose intolerance, celiac disease, chronic constipation, chronic pancreatitis or other malabsorption disorder);
Presence of mental health disorders (including (a) lifetime history of schizophrenia, schizoaffective disorder, or bipolar disorder, (b) current major depressive episode or a major depressive episode within the last two years, and /or (c) current anxiety disorder or an anxiety disorder in the last 2 years);
Compromised immune system as a consequence of a diagnosis resulting in regular use of steroids or other immunosuppression medications, or thyroid treatment, or use of GLP-1 agonists.
Who will take informed consent? < 26a >
Eligible participants will receive consent documents from the trial coordinator prior to Visit 1. At Visit 1, informed consent will be confirmed prior to baseline data collection. Participants were made aware that consent can be withdrawn anytime, for any reason.
Additional consent provisions for collection and use of participant data and biological specimens < 26b >
We will indefinitely store any remaining blood and stool samples in a secured -80°C freezer at Monash University’s Hypertension Research Laboratory at the Victorian Heart Hospital. We may reanalyse these samples as new techniques and information emerge. These samples will be coded with a study and participant number, and a log will be kept linking the study sample to the participant’s identity. This log will be kept in a secure area that only the study staff members can access. Participants will be asked to consent to have their information used for this study and future related research.
Interventions
Explanation for the choice of comparators < 6b >
This study is a placebo-controlled crossover study. As such, changes before and after the intervention will be calculated within each participant and between placebo and intervention groups, shown as placebo-subtracted effects.
Intervention description < 11a >
Study setup, data collection, and analyses
Eligible participants will attend four visits at the Alfred Centre over a 48-day period (Fig. 1). Screening will be conducted via REDCap or phone interview. Those eligible will receive consent documents and sample collection kits prior to Visit 1, where informed consent will be confirmed and baseline data collected, including MSNA, ECG, continuous non-invasive blood pressure (NIBP) and respiration, blood samples, faecal samples, urine samples, cognitive tests, and questionnaires. Participants will be randomised to receive either the intervention or placebo shake, with continuous heart rate and 24-h blood pressure monitoring. Stool and urine samples, food diaries, and gastrointestinal transit tests data will be collected throughout. Visits 2 and 3 will repeat key assessment post-intervention and pre-second phase, respectively, with participants switching arms after a washout period. Visit 4, will mirror Visit 2, including collection of biological samples, recordings of MSNA, ECG, NIBP and respiration, cognitive testing, and 24-h monitoring. Home assessments after each visit include consuming a blue muffin to assess gut transit time, returning ABPM monitors, recording daily BP, wearing heart rate monitor, and logging supplement intake.
Home sampling, storage, and transportation of biological samples
Faecal and urine samples are self-collected at home using provided kits and immediately stored at -20 °C in the participant’s freezer. At each study visit, participants transport the frozen samples to the study site in the cooler bag with freezer packs. Upon arrival, samples are promptly transferred to -80 °C storage. New collection kits are distributed to participants during visits 1 to 3 for use in subsequent sample collections.
Intervention and placebo shake formulation and sensory evaluation
The intervention and placebo shakes were developed in collaboration with a Melbourne‑based protein shake company, Premium Blends. The intervention shake contains 20 g of HAMSA/B (Ingredion) along with approximately 5 g of additional ingredients: xanthan gum, natural flavour, guar gum, citric acid, malic acid, steviol glycosides, and natural colours. The placebo shake contains 20 g of base powder and 3 g of additional ingredients: natural flavour, xanthan gum, citric acid, malic acid, steviol glycosides, and natural colours. Sensory evaluation of the shakes was performed to assess acceptability and on average, participants were unable to distinguish between the placebo and HAMSA/B shakes across both flavours.
Measurements and outcome measures
All measurements and timepoints are summarised in Table 1. All outcomes will be analysed as the change from baseline (visit 1) and end of each intervention period (either visit 2 or 4). Unless otherwise specified, outcomes will be aggregated using mean values.
Table 1.
Data collection timepoints
| Visit 1 | Between visits | Visit 2 | Visit 3 | Between visits | Visit 4 | |
|---|---|---|---|---|---|---|
| Clinic | ||||||
| Office BP | x | x | x | x | ||
| 24 h-BP | x | x | x | x | ||
| Anthropometry | x | x | x | x | ||
| Blood collection | x | x | x | x | ||
| Finapress BP | x | |||||
| ECG | x | x | x | x | ||
| MSNA | x | x | x | |||
| Cognition | x | x | x | x | ||
| Questionnaires | ||||||
| VAS for irritable bowel syndrome | x | x | x | x | ||
| Profile of mood scale | x | x | x | x | ||
| QoL | x | x | x | x | ||
| Pittsburgh sleep quality index | x | x | x | x | ||
| Gastrointestinal symptom rating scale | x | x | x | x | ||
| Plastic-associated chemical exposure | x | x | x | x | ||
| At Home | ||||||
| Stool collection | x | x | x | x | x | x |
| Bristol Stool charting | x | x | x | x | ||
| Urine collection | x | x | x | x | ||
| 3-day food diary | x | x | x | x | ||
| Home BP | x | x | ||||
| Shake intake diary | x | x | ||||
| Palatability questionnaire | x | x | ||||
| Study review questionnaire | x |
Primary outcomes
MSNA
Microelectrodes inserted into muscle fascicles of the common peroneal nerve will be used to quantify muscle sympathetic nerve activity using standard approaches in Prof Macefield’s Human Autonomic Neurophysiology Lab at The Alfred Centre. As noted above, MSNA is greatly elevated in hypertensive patients [18]. In addition, ECG will be recorded with standard chest leads, continuous non-invasive blood pressure recorded from the fingers (Nova, Finapres Medical Systems, The Netherlands) and respiration recorded by a respiratory belt transducer around the lower chest or abdomen. These recordings will be taken during visits 1, 2 and 4. These measurements will be collected by Prof Macefield and his team at the Alfred Centre as outlined previously [19].
ECG recordings
Heart rate variability (HRV), obtained from ECG recordings via surface electrodes applied to the chest, coupled with respiratory monitoring, will be used as a proxy measure of cardiac vagal nerve activity. short-term or high frequency HRV is due to changes in cardiac vagal activity, and decreases in these indices are associated with poor health outcomes. Recordings will be taken during visit 1–4. These measurements will be collected by qualified researchers at the Alfred Center.
Neurovascular transduction
Sympathetic neurovascular transduction will be computed by measuring the degree of vasoconstriction, reflected in the increases in blood pressure, produced by bursts of muscle sympathetic nerve activity (MSNA). This metric will be obtained using the Ensemble software package (Elucimed, New Zealand).
Sympathetic baroreflex sensitivity
Spontaneous sympathetic baroreflex sensitivity measures the inverse relationship between the beat-to-beat fluctuations in diastolic pressure and muscle sympathetic nerve activity (MSNA) burst incidence, and will be calculated using the Ensemble software package.
Cardiac baroreflex sensitivity
Spontaneous cardiac baroreflex sensitivity measures the direct relationship between the beat-to-beat fluctuations in systolic pressure and cardiac interval, and will be calculated using the Ensemble software package.
Heart rate variability
Time-based and spectral components of heart rate variability (HRV) will be obtained from the short-term ECG recordings obtained in the laboratory with the MSNA, sympathetic nerve activity (MSNA), BP, and respiratory signals. Time-based (RMSSD) or high frequency (HF) HRV will be used as proxy markers of fluctuations in cardiac vagal outflow, given that these signals primarily reflect fast events mediated by rapidly-conducting myelinated axons directed to the sinoatrial node of the heart [20]. Decreases in RMSSD and HF HRVi are associated with poor health outcomes. Recordings will be taken during visit 1–4. These measurements will be collected by qualified researchers at the Alfred Center.
Secondary outcomes
Blood
A trained phlebotomist will collect ~ 30mls of whole blood in EDTA and SSTII tubes.
Blood will be used to measure some or all of the following:
SCFA and other gut metabolites: performed by gas chromatography and mass spectrometry (Monash University).
Gut permeability biomarkers: plasma lipopolysaccharide binding protein and soluble CD14 (measured by ELISA), and quantification of micro- and nano-plastics.
Peripheral blood mononuclear cells (PBMCs) will be collected for assessment of immune cell function, in conjunction with plasma cytokines (IL1B, IL6, IL10, IL17A, IFN-γ, TNFα, IL21, IL22), all performed at the Marques Lab.
Renal and liver function tests (Alfred Heart Pathology).
Fasting blood glucose and cholesterol (Alfred Heart Pathology).
Faecal samples
Participants will receive a kit to collect a faecal sample at home. The kit will include 4 tubes for sample collections (2 empty, 1 with glycerol, and one with RNA/DNA shield). They will be asked to store the sample at -20 °C until it can be returned to the researchers. Faecal samples will be used to measure some or all of the following:
Microbiome DNA sequencing: gut microbiome will be determined by 16S rRNA (bacteria) or shotgun metagenome sequencing (for other microbes).
Flow cytometry: To quantify bacterial load.
Markers of gut inflammation: Faecal calprotectin, faecal lipocalin-2
SCFA and other gut metabolites: performed by gas chromatography and mass spectrometry.
Faecal samples will be collected 1–3 days before all visits.
Urine samples
Participants will receive urine collection kits at each visit for use at their subsequent visit, and kits will also be mailed to them prior to their first visit. On the morning of each visit, participants will collect a midstream urine sample and transport it to the Alfred Hospital, secured in a double-contained biological hazard bag within a cooler bag. Samples will be kept on ice for a maximum of 5 h before being stored at –80 °C. The samples will be used in a future study to examine changes to the kidney transcriptome [21] and BP in relation to SCFA intake.
Office blood pressure
At all visits, office BP will be monitored using an automated digital BP monitor (Omron Healthcare, Japan, HEM-907). After 5 min of resting, the investigator will set up the participants with an automatic BP monitor that will take 3 automated measurements while the researcher is not in the room (to limit the white coat effect).
Ambulatory blood pressure monitoring
At the end of all visits, participants will be set up with a 24-h ABPM monitor, Mobil-O-Graph (I.E.M GmbH), and given instructions to complete a diary and what activities to avoid. ABPM involves readings every 15–30 min over 24 h, which generate whole-day, daytime, and nighttime BP.
Home blood pressure monitoring
At visits 1 and 3, participants will be asked to record their BP twice daily (in the morning and afternoon) during the intervention periods.
Gastrointestinal transit time
The blue muffin experiment aims to measure the extent of gut transit time in hypertension and with the intervention, as a key determinant of the gut microbiome [22, 23]. Participants will be provided muffins made with blue food dye at visits 1–4. The participants will be asked to consume the muffin during each visit, or on the morning after each visit to break their fast. Participants who have gluten intolerance will be given the blue food dye and asked to consume it with a breakfast option. We will provide instructions to note the time the muffin was consumed and the time it was excreted (documented by noting the appearance of blue stool). Muffins are prepared in a HACCP certified facility (i.e., BASE Kitchen at Monash Department of Food, Nutrition and Dietetics) by research team members who have completed the Do Food Safely training program from the Department of Health, Victoria. Muffins will be vacuum-packed, labelled with a best-before date and frozen.
Profile of Mood Scale (POMS)
The POMS, consisting of 65 items, was designed to assess individuals across seven mood domains: fatigue-inertia, anger-hostility, vigor-activity, confusion-bewilderment, depression-dejection, tension-anxiety, and friendliness [24]. This scale is recommended for monitoring changes during a short-term treatment or assessment period; therefore, it is well suited for the GRAINS-BP trial.
Assessment of Quality of Life-4D (AQoL-4D)
The AQoL-4D measures an individual's overall well-being, including a physical pain sub score [25]. It includes 35 items covering happiness, pain, self-worth, coping, and relationships. Participants are asked to select the option that most accurately reflects their experiences over the past week, with choices ranging from "never" to "always.” Per GCP guidelines, participants will be asked for their consent in the consent form to complete this assessment.
Visual Analogue Scale for Irritable Bowel Syndrome (VAS-IBS)
The VAS-IBS is a self-report scale that evaluates the severity of symptoms related to irritable bowel syndrome [26]. It uses a 10-cm scale, where participants mark the point that best represents the intensity of their symptoms, ranging from "no discomfort" to "worst possible discomfort".
Plastic-Associated Chemical Exposure Questionnaire (PACeQ)
The PACeQ questionnaire [27] will be used to measure the likely exposure to sources of plastics-associated chemicals found in the participants food, personal care products and the surrounding environment. It’s a three-part questionnaire, focusing on plastic-associated chemical consumed within the last 24-h.
Pittsburgh Sleep Quality Index (PSQI)
The PSQI is a self-report questionnaire developed at the University of Pittsburgh, designed to assess sleep quality, latency, duration, efficacy, disturbances, use of medication, and daytime dysfunction [28]. The results can be analysed to distinguish between poor sleepers and good-quality sleepers.
The Gastrointestinal Symptom Rating Scale (GSRS)
The GSRS measures abdominal pain, reflux, indigestion, diarrhoea, and constipation [29]. The Likert scale is used to quantify the intensity of gastrointestinal symptoms and produce a score on the impact of daily life. The GSRS is evaluated and widely used in clinical studies to monitor dietary and medicinal intervention outcomes.
Cognitive tests
We will use five cognitive tests from the NIH Toolbox. The NIH Toolbox provides short tests designed to measure mental processes such as remembering, judging, and problem-solving. The tests assess functions such as pattern recognition, short-term recall, and logical reasoning. We will use the (1) Flanker Inhibitory Control and Attention Test [30], (2) List Sorting Working Memory Test [31], (3) Pattern Comparison Processing Speed Test [32], (4) Picture Sequence Memory Test [16], and (5) Dimensional Change Card Sort Test [33].
Bristol stool charting
The Bristol Stool Chart is a diagnostic tool for self-rating stool types based on their appearance [34]. The scale includes seven distinct categories, from hard, pellet-like stools to liquid stools, allowing for an assessment of bowel function and potential digestive health issues.
Dietary assessment
Dietary intake will be assessed using 3-day food diaries, including two weekdays and one weekend day, analyzed using FoodWorks Professional Software (Xyris). Participants will be asked to download the Easy Diet Diary app (compatible with both iOS and Android) and shown how to record their food consumption.
Criteria for discontinuing or modifying allocated interventions < 11b >
If the participant’s home BP increases to systolic BP > 160 and/or diastolic BP > 100 mmHg, we will encourage the participants to withdraw from the trial and seek medical attention.
Strategies to improve adherence to interventions < 11c >
Adherence to the intervention will be monitored through the return of unconsumed shake sachets at the end of each intervention arm. Participants will also record when shakes are consumed in their shake intake diary.
Relevant concomitant care permitted or prohibited during the trial < 11d >
In addition to the exclusion criteria, participants will be asked not to attempt to lose weight and to limit their intake of foods high in resistant starches/SCFAs during the trial:
• Kombucha (fermented tea).
• Vinegars: Apple cider vinegar, white/red wine vinegar, white vinegar, malt vinegar.
• Pickled foods: pickled onion, pickles (also known as pickled cucumber, or gherkins).
• Sauerkraut.
• Tomato sauce.
• Tabasco sauce.
• Kimchi.
• Soft cheeses (particularly blue and brie).
• Kefir.
• Products containing BarleyMax™
• Under ripe bananas (quite green).
• Green Banana Flour.
• Waxy Maize starch.
Provisions for post-trial care < 30 >
Monash University’s insurance policy will provide trial participants coverage in case of any injury or harm resulting from trial participation. All participants will be informed of the study's potential risks during the informed consent process, including a discussion of the procedures to ensure their safety during the trial.
Participants will be offered appropriate medical care at no cost if any harm or injury occurs due to trial participation. This care will be provided through the Alfred Hospital or another appropriate healthcare provider, as determined by the trial team in consultation with medical professionals.
Outcomes < 12 >
The primary outcome of this study is changes in autonomic nervous system (ANS) outflow. MSNA will be measured directly by microneurography, while changes in the magnitude of cyclical parasympathetic drive to the heart will be assessed indirectly using heart rate variability indices generated using ECG recordings. Changes in ANS activity before and after the intervention will be calculated within each participant and between placebo and intervention groups, and shown as placebo-subtracted effects.
The secondary outcome of this study is changes in 24-h BP, gut microbiome, SCFAs, immune cells, gastrointestinal transit time, quality of life, symptoms of anxiety and depression, cognitive function, gut barrier markers, and circulating microplastics.
Participant timeline < 13 >
Refer to the study intervention description section, Fig. 1, and Table 1 above.
Sample size < 14 >
There is no preliminary data available on SCFAs and changes to ANS; thus, we used our previous data on BP [10] to estimate sample size. Using PASS (version 24.0.2) with the support of a statistician, Dr Tim Powers (Monash University statistics consultant) and a 2 × 2 cross-over design, in this proof-of-principle study, we estimated we would require 24 participants to achieve 80% power with α = 0.05 (calculated effect size 0.5) to determine a 5-mmHg difference in 24-h SBP after intake of SCFAs for 14 days based on our previous clinical trial [10]. Although this effect size is derived from results referring to changes in blood pressure, we note that changes in blood pressure is not the primary outcome in this trial. This primary outcome examined here is ANS activity in the context of hypertension, for which no preliminary data exists. Therefore blood pressure was used as a proxy to inform sample size estimation in this trial. We applied a conservative correlation coefficient of intraperson correlation of ρ = 0.5, consistent with value commonly reported in crossover BP studies. We will recruit 29 participants in total to account for a 20% dropout rate.
Recruitment < 15 >
We will combine several recruitment strategies. This will include using our clinical database (n > 200), where participants have given approval to be contacted about new research, public campaigns to measure BP in the community (e.g., May Measurement Month, where BP is measured in public spaces), as well as advertising and interviews in traditional media, online and social media (Monash website, Marques Lab website, Facebook groups and ads, Twitter, LinkedIn, Instagram, Google ads etc.). We will also have advertising material in public places (e.g., sports clubs, senior citizens clubs, churches, local councils, shopping centres, GP offices) and clinics. Patients who attend hypertension specialist clinics at the Alfred Hospital Heart Centre, and who fit the selection criteria will be offered to places in the study. If they give verbal or written consent to participate in further research, their name and contact details will be shared with the researchers, who will contact them directly. Interested participants will be directed to the Hypertension lab website (https://www.marqueslab.com/trials) where there is a link to the initial screening questionnaire. Alternatively, interested participants may contact the researchers through the number or email advertised on the flyer and the website. The trial also gained exposure through an article in a Melbourne-based tabloid (The Herald Sun) as well as nationally syndicated Channel 7 and 9 News.
Assignment of interventions: allocation
Sequence generation and concealment mechanism < 16a, b >
The allocation procedure for this trial is identical to our previous clinical trial [10]. The allocation sequence was generated externally by the principal investigator using computer generated random numbers. A 1:1 allocation ratio was used with stratification by age, sex and BMI. The stratified block randomisation list was then uploaded onto REDCap as a pre-prepared allocation table. REDCap was not used to generate the randomisation sequence, only to implement the uploaded list. REDCap reveals the participant’s allocation only when the randomisation button is clicked for each participant, ensuring that the allocation remains concealed until enrolment is complete. The trial coordinator will use REDCap to randomly allocate participants to either twice daily HAMSA/B or placebo (waxy maize-starch) shake. Both the study participants and coordinator are blinded to the interventions. Participants will be assigned random participant codes and only this information and their visit number will be available on data forms and files.
Implementation < 16c >
Participants will be enrolled by the study coordinator, who confirms eligibility and completes the enrolment procedures. After enrolment, the coordinator enters the participant’s information into REDCap. Randomisation is then performed by clicking the REDCap randomisation button, which reveals the participant’s allocation. The study coordinator therefore receives the allocation directly from REDCap and provides the participant with HAMSA/B or placebo (waxy maize-starch) shakes accordingly. The allocation sequence itself was generated by the principal investigator and uploaded into REDCap as a pre-prepared allocation table, Future assignments and the block structure are not visible at the point of randomisation, and only the allocation revealed when the randomisation button is pressed is used for assigning participants.
Assignment of interventions: blinding
Who will be blinded < 17a >
The participants, trial coordinator, and investigators involved in data collection are blinded to the intervention group. Data analysts will also be blinded to the intervention group. The HAMSA/B and placebo shakes will be made by a Melbourne-based food and sports supplement manufacturer. These shakes will be aliquoted into 40g doses in sealed sachets, labeled as A and B for blinding purposes. Prof Marques will be the only investigator that remain unblinded throughout the trial.
Procedure for unblinding < 17b >
Unblinding will only occur if a participant feels unwell and needs immediate medical attention or when we reach at least n = 10 for interim analysis.
Data collection and management
Plans for assessment and collection of outcomes < 18a >
All investigators involved in data collection analysis are either trained or will be trained in the respective methods. Only reliable and validated methods are used in this study. References to data collection can be found in this protocol.
Plans to promote participant retention and complete follow-up < 18b >
The trial coordinator will ensure detailed study information is provided to the participants and informed consent is obtained with clear expectations. The trial coordinator will send out reminder texts and regular check-ins to maintain engagement. Participants will be offered some flexibility in regard to start dates and visit dates. Participants will be reimbursed AUD $400 at the end of the trial. The reimbursement amount was calculated by matching the minimum wage in Victoria (AUD $24.10) multiplying by the total number of hours required for the trial (16 h) and rounding up. Participants will also be provided with vouchers to cover their parking and/or public transport costs.
Data management < 19 > and Confidentiality < 27 >
All data will be maintained as de-identified. Data will be stored in REDCap or a secured Monash Google Drive with limited access. Paper files will be maintained in a locked storeroom at the Victorian Heart Hospital with restricted access. Personal information about potential and enrolled participants will be handled with the utmost care to maintain confidentiality throughout the trial. At the beginning of the study, the explanatory statement will outline how personal data will be collected, used, stored, and protected, in compliance with relevant privacy laws and ethical guidelines. Only necessary demographic and health information will be collected during participant recruitment, and all personal data will be stored securely in password-protected files or locked cabinets. Throughout the trial, identifying information will be replaced with unique participant codes to ensure that personal data is not linked to any study results. The relevant investigators will undergo training in confidentiality protocols, and access to personal data will be restricted to authorized personnel only.
Participants’ identities will not be disclosed when sharing data for analysis or reporting. After the trial, any personal information retained will be used to potentially participate in an upcoming trial with the participants’ consent. Participants will be informed of their right to withdraw consent at any time, and all data associated with withdrawn participants will be removed or anonymized as required. Only the researchers listed as authors on this paper will have access to the trial data. Access to the final trial dataset will be restricted to authorized personnel only. Primary access will be granted to the study coordinator and the study’s data management team for data analysis, interpretation, and reporting. Other members of the research team may be granted access as needed based on their role in the study. All data dissemination will be in line with ethical standards and regulations regarding research data.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use < 33 >
All biological samples will be processed and stored in a secured -80 °C freezer at Prof Vaughn Macefield’s Laboratory and the Alfred Hospital Learning Centre, and permanently stored at the Monash University’s Hypertension Research Laboratory (Marques’ lab) at the Victorian Heart Hospital. All biological samples will be coded and will not contain any direct identification of the participants.
Statistical methods
Statistical methods for outcomes < 20a >
To evaluate the change in nerve activity within each participant, we will perform post-hoc comparisons to analyse the pre- and post-intervention nerve activity at each visit and general linear mix (GLM) models with repeated measures between and within participants. Data will be adjusted for sex, BMI, age, and study arm (intervention sequence) to account for potential carryover effects from the washout period, ensuring an accurate comparison between interventions. Since we will perform multiple comparisons, we will use a Šidák correction to control the family-wise error rate.
As for the secondary outcomes, we will conduct GLM and multiple regression analyses to assess how various factors influence changes in ANS outflow, by quantifying the relationship between predictors (changes in microbiome, immune cells, difference in questionnaire answers, and gastrointestinal transit time) and the outcome (changes in BP, ANS outflow).
Interim analyses < 21b >
A single interim analysis will be performed in August 2026 by a study statistician with oversight from the primary investigator. The purpose of the interim analysis will be to examine data-quality, not for hypothesis testing or stopping rules. The study coordinator will receive only unblinded, anonymised output. Please see Supplementary File for detailed statistical analysis plan.
Methods for additional analyses < 20b >
Data will be adjusted for sex, BMI, age, and study arm.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data < 20c >
All analyses will be conducted using the intention-to-treat (ITT) principle, whereby all participants will be analyzed in the groups to which they were originally assigned, regardless of adherence to the intervention protocol or withdrawal from the study.
Missing data will be handled using multiple imputation under the assumption that data are missing at random. The imputation model will include baseline covariates, intervention sequence, and available outcome data. Estimates and standard errors will be combined across imputed datasets using Rubin’s rules, which will account for both within- and between-imputation variability. Sensitivity analyses using complete-case data will be conducted to assess the robustness of the primary and secondary outcome findings.
Plans to give access to the full protocol, participant level-data and statistical code < 31c >
Participant-level data and statistical code will be shared through a data-sharing agreement, allowing researchers to request access for scientific or educational purposes. Procedures for accessing this data will be outlined in a data-sharing policy, which will be developed in compliance with applicable privacy regulations and institutional guidelines.
Oversight and monitoring
Composition of the coordinating centre and trial steering committee < 5d >
The trial coordinator will meet with the principal investigators fortnightly to discuss progress. Ad hoc meetings will be scheduled for the trial steering committee at significant milestones, to review data, or address urgent issues.
Composition of the data monitoring committee, its role and reporting structure < 21a >
A data monitoring committee is not required for this trial given that it is a short-duration trial with minimal risk—this is a supplement-based trial in which the product has been tested and is safe for human consumption. Instead, Prof. Marques, who will remain unblinded and independent of data collection and analysis, will perform the interim analyses and monitor the safety and endpoint data.
Adverse event reporting and harms < 22 >
Adverse events will be reported to the Monash University Human Research Ethics Committee. No adverse events were reported with the HAMSA/B supplement in our previous trial [10]. Harm will be assessed through participant self-reporting at each study visit and through objective monitoring of renal function, including serum creatinine and estimated glomerular filtration rate (eGFR), performed through Alfred Pathology, to confirm no adverse effects on kidney function. All harms will be coded and reported using MedDRA (Medical Dictionary for Regulatory Activities) standardised terminology.
If any incidental findings are identified during the study, the following process will apply. For abnormal ECG results from the myPatch Holter or ABPM data, clinical cardiologists who are investigators in this study will be consulted. The participant will then be contacted and advised to follow up with their GP, along with a referral letter. Similarly, if abnormal pathology results are reported, the participant will be notified and provided with a referral letter for GP consultation.
Frequency and plans for auditing trial conduct < 23 >
The trial may be subjected to internal and external audits as directed by the Monash Human Ethics Research Committee, independent from the investigators.
Plans for communicating important protocol amendments to relevant parties < 25 >
Any significant modifications to the trial protocol—including changes to eligibility criteria, primary or secondary outcomes, statistical analysis methods, or other core design elements—will be promptly communicated to all relevant parties. This includes formal submission to the institutional Human Research Ethics Committee (HREC) for review and approval prior to implementation. All investigators and trial staff will be notified through written correspondence or team meetings to ensure consistent application of protocol updates.
Trial participants will be informed of any changes that may affect their participation, safety, or consent, and re-consent will be sought where appropriate. Updates will also be made to the trial registration record with ANZCTR to ensure public transparency. If applicable, significant amendments will be disclosed in publications or communicated to journals where the trial is registered or published. Regulatory authorities will be notified if required.
Dissemination plans < 31a >
Upon completion of the trial, participants, healthcare professionals, the public, and other relevant groups will receive the results through journal publications. The trial results will be submitted to peer-reviewed journals for publication and presented at conferences. There will be no restrictions on the publication of the trial results unless required by applicable regulatory agencies or agreements with collaborating entities, such as funding bodies. Even if the results are negative or inconclusive, they will be published to contribute to the broader scientific knowledge base. Authors will be selected based on their substantial contributions to the study’s conception, design, data acquisition, analysis, interpretation, and drafting or revising the publication according to authorship guidelines as per our lab manual, ICMJE, and NHMRC guidelines [35–37]. The final manuscript will be reviewed and approved by all authors before submission, ensuring the trial investigators' active participation in reporting results.
Following the trial’s conclusion and in accordance with ethical and regulatory guidelines, we plan to make the participant-level data and statistical code publicly accessible. The protocol and results will be made available in scientific journals through trial registries and, where appropriate.
Discussion
Research into the gut microbiome has revealed a new avenue for treating many health conditions, including cardiovascular diseases. One of the major breakthroughs in the gut microbiome field was the identification of SCFAs as gut microbiome-derived metabolites that confer health benefits in many diseases, including hypertension. Despite their positive effects, the underlying mechanisms of how they confer protection remain unclear. Current studies point to the involvement of neurotransmitters and immune modulation. In the context of hypertension, we believe that SCFAs affect blood pressure regulation by influencing the multifaceted gut-brain-immune axis, specifically bringing about their effects by reducing muscle sympathetic nerve activity. GRAINS-BP will shed light on these mechanisms; by understanding how SCFAs confer their protective effects, new therapeutics could be developed for cardiometabolic and other conditions.
Trial status
GRAINS-BP is currently recruiting.
Supplementary Information
Acknowledgements
We would like to thank the Hypertension Research Laboratory’s Consumer Advisory Panel for their feedback on the trial design. We would also like to thank Dr Tim Powers, Monash University, for his statistical guidance.
Abbreviations
- BP
Blood pressure
- MSNA
Muscle sympathetic nerve activity
- HAMSA/B
High-amylose maize starch acetylated/butyrylated
- ECG
Electrocardiogram
- SCFA
Short-chain fatty acids
- GLM
General linear model
Authors’ contributions < 31b >
Conceived and designed the study: RRM, FZM. Supervised the study: RRM, FZM, VGM. Contributed to data or analysis tools: DR, BM, JSA, DF, CY, JL, MS, PC, MP, TM, SD, ED, EL, VGM. Wrote the paper: MW, FZM, RRM. Reviewed the paper: all authors. Secured funding: FZM, RRM. All authors read and approved the final manuscript.
Funding < 4 >
This study is supported by a NHMRC Investigator Grant (GNT2017382) and Senior Medical Research Fellowship from the Sylvia and Charles Viertel Foundation awarded to Prof Francine Marques and a Foundation for High Blood Pressure Research grant awarded to Dr Rikeish R. Muralitharan.
Data availability < 29 >
All data and materials will be made available either through clinical registries and/or scientific journals where appropriate. Participant-level data and statistical code will be shared through a data-sharing agreement, allowing researchers to request access for scientific or educational purposes. Procedures for accessing this data will be outlined in a data-sharing policy, which will be developed in compliance with applicable privacy regulations and institutional guidelines.
Declarations
Ethics approval and consent to participate < 24 >
Ethics approval has been provided by the Monash Human Ethics Research Committee (reference number: 46683). Written, informed consent to participate will be obtained from all participants.
Consent for publication < 32 >
Not applicable. No details, images or videos relating to any individual has been included in this manuscript.
Competing interests < 28 >
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Francine Z. Marques and Rikeish R Muralitharan are joint senior authors.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All data and materials will be made available either through clinical registries and/or scientific journals where appropriate. Participant-level data and statistical code will be shared through a data-sharing agreement, allowing researchers to request access for scientific or educational purposes. Procedures for accessing this data will be outlined in a data-sharing policy, which will be developed in compliance with applicable privacy regulations and institutional guidelines.