The purpose of this study is to quantify the acute effects of an acid-active protease enzyme supplement (P24) on postprandial aminoacidemia following consumption of a standardized mixed-protein meal containing both plant- and animal-derived protein sources. Secondary objectives include evaluating the postprandial plasma peptidomic response, characterizing the postprandial hormonal responses (GLP-1, insulin, and GIP), and assessing the postprandial glucose response using standardized capillary blood glucose measurements. The study will also evaluate subjective responses including satiety and hunger and gastrointestinal symptoms.

This study is a randomized, double-blind, placebo-controlled crossover trial designed to evaluate the acute effects of an acid-active protease enzyme supplement (P24) on postprandial amino acid availability after consumption of a standardized mixed-protein meal in healthy adults. Each participant will complete two morning test visits separated by an approximately 7-day washout period and will receive P24 during one visit and placebo during the other visit in randomized order. Each participant will therefore serve as his or her own control.

The primary objective is to compare postprandial circulating total amino acid responses between the P24 and placebo conditions following the test meal. Secondary objectives are to evaluate essential amino acids, branched-chain amino acids, postprandial glucose responses, postprandial hormonal responses including GLP-1, GIP, and insulin, plasma peptide profiles, and subjective responses such as hunger, satiety, and gastrointestinal symptoms.

Visits will be conducted in the morning after an overnight fast. Pre-visit standardization will include restrictions on vigorous exercise, alcohol, nicotine, and caffeine prior to each test visit, as well as instructions to maintain similar dietary intake before each study period. At the first visit, height and weight will be measured. At each visit, participants will complete brief questionnaires related to recent diet and activity, sleep, gastrointestinal symptoms, and subjective appetite.

An indwelling intravenous catheter will be placed in a forearm vein for repeated venous blood collection. Venous blood samples will be collected at baseline and at multiple postprandial timepoints over 180 minutes following meal consumption. Capillary blood glucose will also be assessed at matched timepoints using standardized finger-prick measurements. Participants will remain on site during the postprandial observation period and will be monitored for adverse events and procedure-related symptoms.

The test meal will be a standardized mixed-protein burger meal designed to include both animal-derived and plant-derived protein sources. Meal composition, preparation, and serving procedures will be standardized across visits. Participants will consume the meal with water within a defined time window. On each study day, participants will receive either one P24 capsule or one matching placebo capsule immediately prior to the meal according to the study randomization schedule. Active and placebo products will be identical in appearance to maintain blinding.

Targeted amino acid analysis will be performed using LC-MS/MS to quantify circulating amino acid concentrations across the postprandial period. The primary endpoint will be incremental area under the curve for total amino acids over 0 to 180 minutes. Secondary amino acid outcomes will include essential amino acids, branched-chain amino acids, and selected kinetic parameters including maximum observed concentration and time to maximum concentration. Hormonal outcomes will include GLP-1, GIP, and insulin measured at prespecified timepoints. Glucose outcomes will include postprandial incremental area under the curve and related peak and variability measures based on discrete capillary sampling.

Exploratory plasma peptidomic profiling will be conducted in a subset of participants using aliquots from routine plasma samples collected during study visits, without additional blood collection. Subset selection will be performed using prespecified balancing criteria and will not be based on the magnitude or direction of observed treatment effects.

Data will be analyzed using statistical methods appropriate for a crossover design, including paired or mixed-effects approaches as appropriate for each outcome. Standardized procedures for visit conduct, sample handling, laboratory processing, and data review will be used to support data quality and protocol consistency.

Inclusion Criteria:

• Healthy adults aged 45-75 years
• BMI 18.5-35 kg/m2
• Able to provide informed consent and comply with study procedure
• Willing to consume the standardized mixed-protein test meal

Exclusion Criteria:

• BMI outside of 18.5-35 kg/m2
• Gastrointestinal disorders or malabsorption syndromes that could affect digestion/absorption (e.g., inflammatory bowel disease, celiac disease)
• Diagnosed diabetes
• Current use of medications or supplements that materially affect glucose metabolism or digestion (e.g., GLP-1 receptor agonists, chronic digestive enzyme supplementation)
• Resting blood pressure ≥ 140/90 mmHg
• Self-reported cancer within past 5 years
• Known allergy or intolerance to meal components or capsule ingredients
• Self-reported anemia or other conditions that increase risk from blood draws
• Pregnancy or breastfeeding
• Current smoking or nicotine use, heavy alcohol use (defined as > 5 drinks/day or > 15 drinks/week), or other factors that in the investigator's judgment make participation unsafe

This is a double-blind study. Participants and study staff conducting outcome assessments will be blinded to treatment assignment. P24 and placebo capsules are identical in appearance and are labeled according to a computer-generated randomization schedule prepared by a team member not involved in data collection