The aim of the study is to investigate the effect of non-nutritive sweeteners on glucose regulation. Secondary objectives are to investigate the effect of NNS on gut microbiome, gut hormone secretion, sweet taste sensitivity and preference, and the excretion of NNS in urine.

Sugar intake has increased over the years, simultaneously with the prevalence of overweight and obesity. To decrease sugar intake, consumption of non-nutritive sweeteners (NNS) has increased and opinions on its use remain divided. A fully controlled dietary intervention using a NNS mixture and dose that is similar to real-life intake, is therefore necessary to advance our understanding of the potential effects of NNS on glucose regulation, gut microbiome, and gut hormone secretion under real-life conditions.

The aim of this study is to investigate the effect of non-nutritive sweeteners on glucose regulation. Secondary objectives are to investigate the effect of NNS on gut microbiome, gut hormone secretion, sweet taste sensitivity and preference, and the excretion of NNS in urine.

To do this, a single blind, fully controlled dietary intervention with a cross-over design will be performed. The study will consist of 2x8 weeks of intervention, with a washout of > 6 weeks in between. The study population will be 60 healthy adults aged 45-79, with a BMI between 20-35 kg/m2.

The study will consist of a fully controlled diet of two 8-week intervention periods (2 weeks run-in + 6 weeks intervention). The diets differ in the presence of non-nutritive sweeteners (NNS): NNS-diet versus non-NNS diet, where additional NNS are added to the NNS-diet.

The primary outcome measure will be the within person difference in blood glucose incremental area under the curve following a 75 g glucose tolerance test between the NNS diet and the non-NNS diet. Secondary outcome measures are the effect of NNS on Matsuda index, insulin response, blood lipid profile, and gut hormone secretion will be determined. The effect of NNS on small- and large intestinal microbiome will be investigated by a change in composition, functionality, and short chain fatty acid content. The effect of NNS on sweet taste sensitivity, preference, and liking will be investigated, as well as the excretion of NNS in urine and saliva.

The total time invested by participants is around 85 hours. Participants are restricted for 16 weeks in their eating habits, since they follow a fully controlled diet prepared by the research facility. Participants will visit the research facility in total 38 times. In total, ~ 603 mL of blood will be drawn by experienced nurses over the entire RCT (>6 month period). During DEXA scans, participants will receive a total of about 0.006 mSv of radiation.

Invasive measurements that participants can experience as burden are collection of faecal and urine samples, and finding back the Simba capsules in the stool. Non-invasive measurements for participants are wearing the CGM, wearing the ActiGraph, consuming the blue cakes, and performing sensory tests.

Inclusion Criteria:

• Age 45-79 years;
• BMI of 20-35 kg/m2;
• Having veins suitable for placement of a venflon catheter.

Exclusion Criteria:

• Diseases or prior surgeries affecting the stomach, liver, kidneys or intestines (allowed i.e. appendectomy);
• Cardiovascular diseases (e.g. heart failure) or cancer (e.g. non-invasive skin cancer allowed);
• Diagnosed with type 1 or type 2 diabetes;
• Drug treated thyroid diseases (well substituted hypothyroidism is allowed for inclusion);
• HbA1c level >6.5% (>48 mmol/mol), as measured during the screening visit;
• Anaemia defined as Hb concentrations <8.5 mmol/L for men and <7.5 mmol/L for women via finger prick;
• Regular use of/receiving medication interfering with research outcomes (as judged by research physician), such as use of glucose lowering drugs, insulin, or use of medication that impacts the gastro-intestinal system;
• Use of antibiotics over the last 3 months before study start;
• Donated blood within 2 months prior to the screening;
• Food allergies, intolerances (including lactose/gluten intolerance) for products used in the study design and/or dietary restrictions interfering with the study (including special diets and eating disorders);
• Followed a diet that can interfere with the study outcomes within 1 month prior to the screening (e.g. ketogenic, sugar free, carbohydrate free);
• Not willing to eat all products in the study diet, including eggs and dairy. Vegetarian is possible;
• Not willing to consume non-nutritive sweeteners;
• Not willing to quit the use of supplements that can interfere with the study outcomes (e.g. pre- or probiotics).
• Intention to change the intensity of exercise during the study period or planning to join a intensive sport event (e.g. marathon or triathlon);
• Intention to lose or gain weight;
• Working night shifts regularly;
• Smoking regularly
• Use of soft and/or hard drugs (cannabis included);
• Abuse of alcohol (defined as >14 glasses (women) or >21 glasses (men) of alcoholic beverages per week);
• Being pregnant or lactating or planning to become pregnant;
• Inability to understand study information and/or communicate with staff;
• Participation in another study that involves an intervention within two months prior to the intervention;
• Working or doing a thesis/internship at the division of Human Nutrition & Health, or the Food, Health & Consumer Research group of Wageningen Food & Biobased Research.