This study was performed as an acute, single blind, randomised, controlled, crossover trial with three test meals consisting of bread made with either 100% LBB, 100% RB, or 100% WF. Randomisation was done using RedCap®.

The primary outcome was changes in postprandial glucose (given as 4 h iAUC) in subjects with or without T2D. The secondary outcomes were postprandial changes in insulin, glucagon, triglyceride (TG), FFA, gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) calculated as either iAUC or total AUC (tAUC). Furthermore, questionnaires were completed to evaluate e.g. satiety and fullness, as well as perception scores of the test meals.

The study was registered at ClinicalTrails.gov as NCT04702672.

Adults with and without T2D were recruited via local newspapers and online ads. The study took place at Steno Diabetes Centre Aarhus, Aarhus University Hospital, between February and June 2024. All participants provided written informed consent after receiving oral and written information. Eligibility was determined through physical exams, medical history, and blood tests.

Inclusion criteria without T2D: Adults ( ≥ 18 years) without any form of diabetes. With T2D: Adults ( ≥ 18 years) diagnosed according to IDF criteria, with HbA1c between 6 and 9.3% (42–78 mmol/mol).

Exclusion criteria (both groups): Insulin use, once-weekly GLP-1 agonists, acarbose, significant cardiovascular, kidney, liver, psychiatric, or endocrine conditions, steroid treatment, substance abuse, pregnancy, breastfeeding, or legal incompetence. Stable treatment for hypertension or high cholesterol was permitted.

After a standardised evening meal and an overnight fast (from midnight), the study participants arrived at the clinic at 07.30 AM on all three study days.

Smoking was not allowed during the overnight fast or the study visits. Alcohol consumption was not permitted the day before the study days. Anti-hypertensive, cholesterol-lowering and anti-diabetic drugs were paused 24 h before every study day. The three intervention days were separated by a six-day minimum washout.

Antihypertensive medications were temporarily discontinued due to their potential confounding effects on blood glucose regulation, although blood pressure was not an outcome measure in this study. The half-lives of metformin and simvastatin - used by the majority of study participants - are approximately 2–4 h, while those of SGLT-2 inhibitors, DPP-4 inhibitors, and atorvastatin range from 12 to 13 h. We acknowledge that a one-day discontinuation may not allow for complete washout of all drugs across there therapeutic classes; however, due to practical considerations, all medications were paused for the same duration.

During the study days, a catheter was placed in a cubital vein for blood sampling. Baseline questionnaires were completed, and blood samples were drawn. At 0 min the test bread was consumed within the next 10 min along with 250 ml of tap water. A bread perception questionnaire was completed within the first 10 min.

During the following 4 h, blood samples were drawn as following: glucose, insulin, and glucagon at –10, 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min; TG, FFA, GLP-1 and GIP at –10, 0, 30, 60, 120, 180, and 240 min. All blood samples were immediately centrifuged at 3000 g for 10 min at 4 °C; thereafter, plasma samples were frozen at –20 °C and the next day stored at –80 °C, except from plasma for glucose measurements, since this was analysed on the study day.

Questionnaires regarding satiety were completed at: 0, 30, 60, 120, 180, and 240 min. At 120 min, an additional 250 ml of tap-water was served.

Regular nude barley (RB, H. vulgare var. nudum PS3) and high-amylose nude barley (LBB, H. vulgare var. nudum LBB) were grown in 2023 at Aarhus University, Flakkebjerg. RB was developed by Agrologica (Mariager, Denmark). Grains from both varieties were milled using a Komo Fidibus 21 (KOMO GmbH, Germany). The LBB variety was bred to lack Starch Branching Enzyme IIa (SBEIIa), resulting in 46.5% amylose content. Its yield, grain weight, and starch granule shape were similar to the original line, as described in detail previously [12]. Wheat bread was made with commercial Manitoba flour (HavneMøllen, Denmark). All three bread types followed similar recipes and were produced by P.A. Andersen Bakery (Vejle, Denmark). Breads were portioned (160 g), sealed, frozen at −20 °C, defrosted overnight before study days, and served unheated.

Participants consumed a standard commercial spaghetti bolognese meal (1750 kJ; 15.4 g fat, 49 g carbs, 18.2 g protein) the night before each study day (Salling Group A/S, Denmark). Extra foods were allowed if intake was measured and replicated across all test days.

Bread analysis were performed on breads prepared similarly to the study breads.

The moisture content was determined by the weight loss after drying in a vacufuge vacuum concentrator from Eppendorf overnight. The total carbohydrate content was measured as the sum of the dietary fibre and starch content. The Megazyme total starch assay kit (K-TSTA-100A, Wicklow, Ireland) was used to determine the total starch content of samples containing resistant starch following the manufacturer’s instructions. This method variant uses dimethyl sulfoxide and a boiling bath, and dissolution in dimethyl sulfoxide at 100 °C is effective for solubilizing all starches in the bread. The dietary fibre content was determined using the Megazyme total fibre assay kit (K-TDFR-200A, Wicklow, Ireland) in accordance with the manufacturer’s instructions. This method variant uses 1 h incubation with heat-stable α-amylase, which is a critical enzymatic digestion step to remove digestible starch components. The resistant starch content was determined using the Megazyme resistant starch assay kit (K-RAPRS, Ireland).

Plasma glucose was measured by enzyme sensor technology Xylem Brand on YSI 2500 or 2900 (YSI Incorporated, Ohio, USA). EDTA-plasma insulin and glucagon were measured with ELISA (insulin no. 10- 1113-01 and glucagon no. 10-1271-01; Mercodia AB, Sweden). Plasma FFA concentrations were measured with enzymatic colorimetric assays by using commercial kits (code 270–7700, Wako Chemicals GmbH, Germany) on the NOVI apparatus (Perkin Elmer, Connecticut, USA). Triglycerides were measured on an Indiko apparatus with quantitative enzymatic methods using commercial kits (REF 981786, Thermo Fisher Scientific, Roskilde, Denmark). GLP-1 and GIP were measured with NL-ELISA techniques (GLP-1 no. 10-1278-01 and GIP no. 10-1258-01; Mercodia AB, Sweden) on the Multimode Plate Reader EnVision (Perkin Elmer, Connecticut, USA).

When consuming the test meal (time 0–10 min), the participants evaluated the looks, texture, taste, and overall liking of the bread. The evaluation consisted of seven boxes rating from the most negative “1; do not like”, over “3; neither/nor” to the most positive “7; like very much”.

Visual analogue scale (VAS) was used to assess hunger, satiety, fullness, desire to eat and prospective consumption of the test breads. VAS consists of a 150 mm line scale with words anchored at each end, expressing the most negative and the most positive rating. The questionnaires were made on paper at time 0, 30, 60, 120, 180 and 240 min a new paper was used for every time point [13, 14]. Results were converted from mm to percentage when results were analysed.

The power calculation was made to detect a difference in our primary outcome (i.e., postprandial glucose response, given as iAUC) of 20% between diets [10]. The number of participants needed to complete the study and achieve a statistical power of 80% was calculated to be 18 subjects with T2D and 20 subjects without T2D (a < 0.05, b = 0.80). A mixed model ANOVA was used to examine the difference between bread types. P < 0.05 was considered statistically significant. Results are given as mean ± 95% confidence interval (CI) in tables and as mean ± SEM in graphs, unless otherwise stated. All statistical calculations were performed with STATA version 18 (StataCorp LP, Texas, USA) and graphical elements were generated using GraphPad Prism 10 (GraphPad Software, Boston, USA).