Population aging is a worldwide phenomenon that constitutes a major public health challenge, as it is linked to increased health care demands for age-related disabilities, such as cognitive impairment [1]. Evidence suggests that the gut-brain axis (GBA) is important for brain health, as well as for cognition and mood regulation [2,3]. Furthermore, dysregulation of this axis is associated with neurodegenerative and neuropsychiatric disorders including dementia, Parkinson disease, and depression [4-6].

Certain features of the GBA also seem to be intricately linked to the function of the immune system [7,8]. For example, gut microbiota aberrations may trigger low-grade systemic inflammation [3,9], a common condition among older adults, which has been suggested to impact brain function and contribute to cognitive decline [10,11]. For example, it has been shown that higher levels of systemic inflammation are specifically associated with lower resting state functional connectivity between brain regions in the default mode network (DMN), a network whose altered functional connectivity has been associated with reduced cognition among older adults [12,13]. Hence, low-grade systemic inflammation and age-related changes of the gut microbiota may be seen as critical factors in the development of cognitive decline in older adults.

Increasing evidence suggests that diet may affect both the gut microbiota and systemic inflammation [9,14]. During aging, appetite, dietary requirements, and changes in dietary behaviors increase the risk of malnourishment [15]. Malnourishment is accompanied with decreases in muscle mass, muscle strength, and bone mineral density, which lowers physical function and increases the risk for frailty [16-18]. Frailty is also coupled with impairments in the immune system and cognitive decline [19,20]. Taken together, strategies to change dietary behaviors in (especially) vulnerable older adults are warranted.

For older adults failing to meet their daily energy requirements, between-meals snacks can be an effective strategy to promote adequate caloric and nutritional intake [21]. However, the readily available commercial options often consist of a rather unhealthy nutritional profile, containing low quality carbohydrates and unhealthy fats and lacking dietary fiber and proteins. Diets high in glucose and fat but low in protein, fiber, and certain micronutrients may contribute to the development of low-grade inflammation [22], whereas anti-inflammatory diets including Mediterranean diets [23,24], the Nordic diet [25], and diets containing prebiotic components [26-28] seem to exert opposite effects. Such anti-inflammatory dietary patterns have also been linked to improved cognitive function in older individuals [29].

Taking these aspects into consideration, new, drinkable, between-meal products (MiniMeal) were designed based on bioprocessed oat bran and chickpea, providing a well-balanced nutritional profile with high-quality protein and mixed-origin soluble and insoluble dietary fiber. Several previous studies have demonstrated the positive impact of oats on the gut microbiota [30,31], satiety [32], blood pressure [33,34], cholesterol, and glucose regulation (European Food Safety Authority Panel on Dietetic Products, Nutrition, and Allergies). Furthermore, oats have been shown to have anti-inflammatory properties [35,36] and improve cognitive function related to both long- and short-term memory [31]. Chickpeas have been shown to reduce total and low-density lipoprotein cholesterol [37,38], improve homeostatic model assessment for insulin resistance index [37], lower inflammation [1,2], and may modulate the gut microbiota [3-5]. Moreover, evidence from preclinical studies supports the potential neuroprotective role of chickpeas, due to their antioxidant and anticholinergic properties [39,40].

The nutritionally well-balanced MiniMeal between-meal products evaluated in this project (a soup and a fruit drink) contain ingredients with the potential to impact all the above-described health outcomes. By targeting risk factors related to malnutrition, gut-microbiota alterations, and systemic inflammation, these products may promote favorable changes in functional brain activity and cognitive performance. This paper outlines the study protocol for a randomized controlled trial in older adults, designed to evaluate the effects of the MiniMeal products on brain activity during a cognitive task as the primary outcome.

The MiniMeal study is a 3-arm, randomized controlled trial that includes a blinded, 2-armed intervention and a nonblinded, no-intervention comparative arm.

The MiniMeal study will be conducted at Örebro University School of Medical Sciences and the Center for Experimental and Biomedical Imaging in Örebro (CEBIO) at the Örebro University Hospital.

Inclusion and exclusion criteria are found in Textbox 1.

A sample of 114 (38 per arm) community-dwelling men and women, 70 years and older of age, will be recruited in Örebro and surrounding areas through newspaper advertisement and advertisements at primary care facilities, local senior homes, and via local senior organizations. Participants from similar previous studies conducted by our research group (Provita: NCT04126330, AnaBio: NCT05801042) will also be contacted and invited to participate. When interested, prospective participants will be screened for eligibility according to the predetermined inclusion and exclusion criteria, as listed in Textbox 1. Potentially eligible participants will then receive oral and written information from the research team regarding the study, associated benefits and risks, and data management. Before participation in the study, written informed consent will be obtained, and participants will be informed regarding their right to withdraw at any moment during the study. Relevant medical history, concomitant medication, and other demographic data (eg, age and sex) will be recorded in a case report form (CRF). A potential participant will only be included when all inclusion criteria are met, and all exclusion criteria are absent.

All involved researchers will be blind to the initial group allocation. However, blinding for the participants allocated to the no-intervention group will not be possible, as no study products will be distributed to them. The 2 intervention arms, however, will remain blinded in terms of allocation for both researchers and participants. Participants included in the intervention group will receive the MiniMeal products, the reference or control group will receive the reference products, and the no-intervention group will receive no study products and will be instructed to maintain their habitual diet. The no-intervention group will therefore not be blinded to group allocation. This may introduce potential differences between groups related to study engagement, expectations, motivation, or behavioral changes during the study period, which will be considered when interpreting comparisons involving the no-intervention group.

An emergency unblinding procedure will be established to allow the investigators (ANH and CB) the option of disclosing a product assigned to any participant if clinical circumstances require such unblinding. Unblinding will be performed by the principal investigators (ANH and CB) if there is a need, for example, to make a medical decision that requires knowledge about the treatment received. Furthermore, if the study participant experiences any kind of serious adverse event (AE) that she or he attributes to the treatment, even if it is just a perception, we will discontinue the study for that participant. In case of disclosure and unblinding of dietary intervention, the participant will be asked to withdraw from further participation in the trial so as to not bias further analysis (ie, not being blind to the study group can affect the analyses unconsciously).

Participants will come to the study center on 7 occasions, 1 time before (screening visit) and 6 times during the study period to perform 1 or more tests and/or examinations. After the screening, visits 1 and 2 will take place at baseline. Visits 3 and 4 will be the mid-study visits in weeks 3 and 6 of the interventional period. Visits 5 and 6 will take place in the last interventional week, that is, week 9. A timeline of study visits can be found in Figure 1, and a SPIRIT schedule of enrollment, interventions, and assessments can be found in Figure 2.

Although the number of studies using magnetic resonance imaging (MRI) to assess the effects of food products on brain activity during a cognitive task in older populations is limited, a few studies can aid in determining an adequate sample size. Boespflug et al [41] conducted a study to examine the effects of fish oil supplementation on older adults (62-80 years) with subjective cognitive impairment and were able to detect differences in blood-oxygen-level-dependent (BOLD) activation during an n-back task with 11 (fish oil) and 10 (placebo) participants in each group. Boespflug et al [42] also conducted a study with 16 older adults to examine the effects of a food supplement on a working memory task, performing the n-back task in healthy older adults. From this sample size, they were able to detect education by age interactions for task-related activity.

A very limited number of studies have used fMRI to assess the effects of food products in older adults [41,42], thus making the determination of an adequate sample size rather difficult. For the primary outcome, the intervention and reference arms will be compared regarding changes in BOLD signal in approximately 20 regions of the brain (implicated in cognitive function and the GBA), with the aim to be able to detect a change with Cohen d=delta/SD=1 (delta difference in mean values between the 2 groups) with 80% power. Counting in a maximum of 10% dropouts, based on a 2-sample t test and working with a Bonferroni-adjusted significance level of 0.05/20, this would require approximately n=36 per group. To account for additional sources of variability inherent to fMRI data in older populations—including motion artifacts, physiological noise, potential nonnormality of BOLD responses, and the possibility of unusable scans—the sample size was conservatively inflated by an additional 5%. Thus, 38 participants will be recruited per arm.

All data will be collected using a CRF. The investigators will capture every part of the clinical data in the CRF. It is the responsibility of the investigators to maintain accurate CRFs to record all observations and data pertinent to the investigation. For instance, when information needs to be modified, the correction should be made, and the original information to be modified should not be erased but overwritten in a way that both the old information and the newly added one are visible. The corrected information will be transcribed by the authorized person next to the previous value, signed or initialed, and dated. Relevant parts of the CRF will be entered into a database, and all physically recorded data will be stored securely in places with limited access. Data management will be in accordance with the Data Management Plan of the School of Medical Sciences at Örebro University. Data will be stored securely at Örebro University servers and only accessible inside Örebro University or through virtual private network from outside the university. Proper folder structure with appropriate read and write access to the data will be used to store project data. Access rights to the project folders will be based on a user’s role within the project.

The MiniMeal study has been approved by the Swedish Ethical Review Authority (identification number (Dnr) 2023-07609-01) and registered at ClinicalTrials.gov (NCT06353984). All participation in the study is voluntary, and all procedures will be conducted according to fundamental ethical principles as outlined in the Declaration of Helsinki (World Medical Association Declaration of Helsinki) and GCPs in Europe (European Medicines Agency, Science Medicines Health), as well as GLPs. The safety, health, well-being, and anonymity of the participants will be prioritized. Prior to the study, participants will provide written informed consent regarding participation and publication of study data. Any important protocol modifications will be subject to an amendment to the Swedish Ethical Review Authority for new approval. Informed consent will be taken by an authorized member of the study team. In case of an amendment in the research protocol, approval will be obtained from the Swedish Ethical Review Authority (Etikprövningsmyndigheten). Collection and preparation of all laboratory samples at the clinical center will be performed in a standardized way according to a written laboratory manual, to GCP and GLP, all ensuring the quality of the procedures. Samples will be stored at the Örebro Hospital Biobank, and all the procedures will be following the Guide to Biobanks in Sweden—Access to Samples for Research and Clinical Trails (Biobank Sverige)—and adhere to the Swedish Biobank in Medical Care Act (SFS 2002:297), of which, all potential participants will be informed in the participant information. All participants will be assigned a unique study ID code, which will be documented and protected by the principal investigator according to good research practice guidelines. Data will be pseudonymized and stored under the assigned study ID code. If necessary, the research team will have access to the code list, but no personal details of participants will be disclosed in any forthcoming publications. Personal information will be anonymized using a participant number. All data will be stored in secure cabinets and backed up on an electronic system with restricted access. Dissemination of findings will take place through presentations or posters at national and international conferences and peer-reviewed publications in high-quality scientific journals, as well as popular science dissemination in regional and national newspapers and outreach activities such as local presentations. All participants receive US $209 after completion of the study.

The primary aim of this study is to investigate whether a nutritionally balanced between-meal product can improve functional brain activity during a cognitive task in healthy older adults. We hypothesize that the 9-week dietary intervention will affect task-related brain activation within predefined regions associated with working memory. In addition, we anticipate that the intervention may positively impact cognitive performance, inflammatory markers, gut function, and physical function, thus impacting several aspects of the GBA. By combining neuroimaging with comprehensive assessments of cognition, gut health, inflammation, and physical function, this study aims to provide novel insights into the mechanism linking nutrition and brain health in older adults.

Given the negative impact of cognitive impairment on older adults’ health and well-being, as well as the increasing demand regarding medical care and associated costs, the primary aim of this study is to investigate the potential benefits of a nutritionally balanced between-meal product on functional brain activity during a cognitive task in older adults. An earlier cross-sectional study by Damoiseaux et al [13] investigated whether functional connectivity of intrinsic brain activity in the DMN is affected by normal aging and found decreased activity in older compared to younger participants. Persson et al [12] investigated the longitudinal changes in DMN while performing a memory task and found a reduced deactivation in DMN regions during advancing age. At the same time, there is evidence from randomized controlled trials that a culturally adapted and individually tailored diet can slow down cognitive aging [67]. Furthermore, evidence indicates that the Mediterranean diet can contribute to improved cognitive function [68].

These previous studies have provided some novel insights into DMN function, as well as linking such measures to systemic inflammation and the importance of a healthy diet. However, to our knowledge, no randomized controlled trials have been conducted to establish any pathophysiological associations. Hence, we aim to measure functional brain activity during a cognitive task by fMRI before and after a 9-week dietary intervention that we hypothesize will positively impact several aspects of the GBA.

As dietary modifications have the potential to modulate both gut microbiota composition and systemic inflammation, they are an attractive option for addressing the aforementioned research questions. However, making long-term, sustainable dietary alterations in older adults proves challenging, given the tendency to revert to former dietary habits over time [69]. Consequently, an alternative strategy involving the addition of healthy between-meal products emerges as an interesting and potentially more sustainable solution for enhancing both the quantitative and qualitative aspects of dietary intake among older adults. Thus, this approach could offer incremental improvements in dietary habits without necessitating drastic changes, thereby promoting long-term adherence and optimizing health outcomes in this population.

The MiniMeal products used in this study contain oat bran and chickpeas, 2 natural food components that have the potential to positively affect gut microbiota and lower inflammation. These 2 areas are of particular interest as they can promote health in older adults including improved brain function. Dietary fibers such as β-glucans in oats, oligosaccharides such as ciceritol in chickpea grains, and the chickpea isoflavone Biochanin A present significant neuroprotective capacity based on preclinical evidence [40,70,71]. Furthermore, intake of additional dietary fiber derived from various sources (grains, legumes, and vegetables or fruits) in the study products has been associated with improved cognitive function and healthy aging [72]. Additionally, the fermentation of these very ingredients in the gut could improve the production of microbial metabolites such as short-chain fatty acids, which may cross the blood-brain barrier and exert their antioxidant and anti-inflammatory properties on the brain with the potential to prevent neurodegeneration [73]. Simultaneously, the extra protein and increased caloric intake also have the potential to prevent malnutrition in individuals at risk and decrease the impact of anabolic resistance [74]. Improving protein synthesis is of particular importance for a healthy bone and muscular system in older individuals. As such, extra caloric intake combined with protein can be seen as an important strategy to fend off frailty [75].

An important strength of the present study is the comprehensive characterization of healthy older adults through multimodal assessments of neuroimaging, cognition, gut health, inflammatory biomarkers, dietary intake, gastrointestinal function, physical function, PA, sleep, and well-being. This broad approach acknowledges the complex interactions within the GBA and may provide valuable mechanistic insight into the pathways through which dietary interventions influence brain health. The inclusion of several secondary and exploratory outcomes is intended to complement the primary neuroimaging outcome and generate hypotheses for future studies while facilitating comparison of the study population with other cohorts.

Nonetheless, designing such a protocol is associated with several challenges. There is currently no universally accepted approach for sample size calculations in functional MRI studies, although considerable methodological advances have been made in recent years [76]. The sample size for the present study was determined based on fMRI-specific calculations together with previous experience from similar studies including adults (aged 18-80 years), as described in the Sample Size and Statistical Analyses sections. Furthermore, although the inclusion of multiple secondary and exploratory outcomes provides a comprehensive assessment of healthy aging, it also increases the complexity of the statistical analyses. To address this, primary, secondary, and exploratory outcomes have been predefined, and appropriate adjustments for multiple comparisons are described in the Statistical Analyses section.

In addition, although task-based fMRI provides a sensitive measure of changes in functional brain activity, there is currently limited evidence regarding the magnitude of BOLD signal changes that should be considered biologically meaningful following nutritional interventions. Given the expected variability of fMRI measures and the relatively short intervention duration, the present study is primarily designed to detect intervention-associated changes and provide estimates of effect sizes rather than establish clinically meaningful thresholds.

Another important consideration is the expected frequency of incidental findings in this study population. As participants aged 70 years and older are included, incidental findings are expected in approximately 5%-20% of MRI scans and around 10% of blood samples [6-8], although fewer than 2% are anticipated to be clinically significant [6]. Brain scans will be acquired by specialized nurses and reviewed by a neuroradiologist. If findings are considered clinically relevant and not attributable to normal aging, participants will be informed and advised to seek further medical evaluation. Prior to enrollment, participants will be informed that the MRI examination is performed for research purposes and is not intended as a clinical screening tool.

Finally, in food intervention trials, the reference product is often designed to be isocaloric to the intervention. In the present study, however, the objective is to determine whether nutritionally balanced between-meal products with higher nutritional quality and energy content than commonly consumed products improve brain health and other indicators of healthy aging. Therefore, the reference soup and drink were designed by Glucanova AB to reflect the average nutritional composition of commonly available tomato soups and fruit drinks on the Swedish market, thereby providing a comparison that more closely reflects real-world dietary choices.

If the intervention demonstrates favorable effects on functional brain activity and related outcomes, this study will potentially provide important mechanistic evidence supporting the role of nutrition in healthy cognitive aging. The findings could then contribute to the development of larger randomized controlled trials and help identify dietary strategies and participant characteristics associated with improved brain health, thereby supporting more personalized nutritional interventions for older adults.

In light of the global increase in cognitive decline among older adults, novel preventive and therapeutic strategies are urgently needed. Greater understanding of both the efficacy of nutritious meals and dietary patterns, and the mechanisms underlying their potential effects on cognitive function, is essential. This paper outlines a randomized controlled trial designed to evaluate the effects of a newly developed, nutritionally balanced between-meal product on functional brain activity during a cognitive task, as the primary outcome. The study is expected to provide new insights into the relationship between dietary habits and cognitive function in older adults and contribute robust evidence from a rigorously designed randomized controlled trial to inform future research. At a societal level, the findings may help clarify the potential of diet-based approaches to support cognitive health and healthy aging in older populations.