*2.4. Study Design*

This was a randomized crossover design whereby each volunteer came for five or nine test sessions with a minimum 1-day wash-out between sessions. The order of the study meals were randomized using the RAND function in Microsoft Excel. Volunteers were asked to avoid strenuous exercise and alcohol intake 24 h prior to their test session and to arrive at the center at 08:30 after an overnight fast of at least 10 h. Upon arrival, volunteers had their baseline (T0) blood pressure measured and a fasted blood sample drawn (after 5 min of rest) at the antecubital vein via cannula by a trained phlebotomist. Volunteers were then served the study meal and instructed to consume the meal within 15 min. Subsequent blood collection was carried out at T15, 30, 45, 60, 90, 120, 150, and 180 min after the first bite. Duplicate blood pressure measurements were taken hourly at T60, 120, and 180 min. A schematic of the study design is shown in Figure 1.



**Figure 1.** Schematic of study design.

#### *2.5. Blood Sampling and Analytical Methods*

Venous blood was collected in K2EDTA vacutainers (BD, New Jersey, NJ, USA), placed immediately on ice, and centrifuged at 1500× *g* for 15 min within an hour of blood draw to obtain plasma. Plasma samples were then stored at −80 ◦C in aliquots until ready for analyses. Plasma glucose and insulin concentrations were determined by the COBAS c311 and e411 automated analyzers (Roche Diagnostics GmbH, Berlin, Germany), respectively. For each analyzer, a successful two-point calibration was performed at the start of the study. Thereafter, quality control was done daily before analyzing the plasma samples. For each assay, 250 μL of thawed undiluted plasma was analyzed in a Hitachi cup using the manufacturer's recommended cassette for glucose and insulin measurement. All samples were analyzed over a 5-day period using the same analytical instrumentations and laboratory consumables (including standards and quality controls). All samples from a given individual (i.e., all treatments and all time points) were analyzed within the same batch and the same day, in order to limit any effects of experimental variations. The inter-batch coefficient of variation for glucose analysis was 7.51%, and that for insulin analysis was 1.99%.

## *2.6. Statistical Analyses*

The primary outcome of this study was the acute effects of the test ingredients on the postprandial glucose and insulin responses for up to 180 min. The secondary outcomes were: i) the effect of the form in which the test ingredients were administered (R vs. D) on the 30, 120, and 180 min postprandial glucose and insulin responses; and ii) the acute effects of the test ingredients on the postprandial blood pressure measured over 180 min. Incremental areas under the curve (iAUCs) of glucose and insulin were calculated using the change in glucose or insulin above baseline fasting concentration, ignoring the area beneath the baseline. Total areas under the curve (tAUCs) of systolic and diastolic blood

pressure measurements were calculated using the absolute blood pressure values measured at the time points.

All analyses in this study were done using Statistical Package for the Social Sciences (SPSS) version 24 (IBM, New York, NY, USA). Data were assessed for normality using the Shapiro–Wilk test as well as visually from the histogram and normal Q–Q (quantile-quantile) plots. Square-root transformation of the data was used where necessary to achieve normality. The treatment e ffect on the iAUCs of insulin and glucose was tested using linear mixed e ffects procedure in SPSS with treatment as the fixed factor and compound symmetry (CS) covariance structure. For the interaction between form and treatment test, a linear mixed e ffects procedure with both treatment and form as fixed factors was used with CS covariance structure.
