To compare postprandial plasma amino acid availability, expressed as incremental area under the curve (iAUC), over a 6 hour period following ingestion of 25 g of different protein isolates in healthy, young adults. The primary objective is divided into the following 2 sub-studies, each with 3 comparisons:

• Sub-study 1: whey protein, calcium caseinate, sodium caseinate.
• Sub-study 2: calcium caseinate, magnesium caseinate, potassium caseinate.

All living tissues are in a constant state of protein turnover, with synthesis and breakdown processes ensuring structural integrity, metabolic flexibility and the capacity for repair. Amino acids are the building blocks of proteins and must be supplied through the diet to maintain a positive protein balance. Ingestion of high-quality protein sources (e.g. meat, dairy) leads to a postprandial increase in circulating amino acids, and as such can facilitate tissue protein synthesis. The magnitude and the kinetics of the amino acid response largely depend on the amino acid composition combined with protein digestion and amino acid absorption rate.

Beyond their structural role, amino acids also function as signalling molecules involved in gastrointestinal hormone secretion and appetite control. One such key hormone is glucagon-like peptide 1 (GLP-1), a gut-derived hormone that plays an essential role in metabolic regulation such as insulin secretion and appetite suppression. Given its role in metabolic health, particularly in the context of obesity and type 2 diabetes, increasing GLP-1 availability has become a target for support in metabolic care. However, many pharmacological approaches to elevate GLP-1 are costly and carry risks of harmful side-effects.

Nutritional strategies that modulate GLP-1 secretion and availability therefore offer an attractive alternative. Emerging evidence suggests that not only protein ingestion itself, but also the matrix in which the protein is delivered can modulate GLP-1 secretion. For example, co- ingestion of specific minerals, especially calcium, has been shown to enhance GLP-1 secretion beyond the effects of protein ingestion alone. These data indicate that mineral-protein interactions may play a meaningful role in appetite regulation and metabolic signalling, warranting further investigation.

Bovine milk contains two primary high-quality protein fractions: whey (~20%) and casein (~80 %). Whey protein is rapidly digested, resulting in fast but transient increases in circulating amino acids, whereas casein protein is digested more slowly, leading to more prolonged but sustained amino acid availability (13-15). To optimize the functional properties of casein, various processing techniques can be applied, including the formation of caseinate salts through binding casein with different minerals such as calcium, sodium, magnesium, and potassium. These caseinate forms differ in mineral composition, which may influence not only digestion and absorption kinetics, but also GLP-1 secretion capacity. Although the differences in digestion kinetics between whey and casein are well-established, a direct comparison of postprandial amino acid and GLP-1 responses following ingestion of whey versus various mineral-bound caseinates has not yet been performed.

To be eligible to participate in this study, a participant must meet all of the following criteria:

• Male or female sex
• Aged between 18 - 35 years inclusive
• BMI between 18 - 30 kg/m2
• Healthy, recreationally active (exercise at least once per two weeks and a maximum of four days per week)
• No physical limitations (i.e., able to perform all activities associated with daily living independently)

A potential participant who meets any of the following criteria will be excluded from participation in this study:

• Intolerant to milk protein and/or dairy-based products
• Smoking regularly
• Diagnosed with gastro-intestinal disorders
• Diagnosed with metabolic disorders (e.g. diabetes)
• Diagnosed with musculoskeletal disorders
• Blood donation in the past 2 months
• Females: pregnancy
• Use of any medication known to affect protein metabolism (e.g. corticosteroids, non-steroidal anti-inflammatories or prescribed acne medications)
• Chronic use of gastric acid suppressing drugs (e.g. proton pump inhibitors, H2-antagonists)

The protein drinks will be prepared by a member of the study team, not involved in the data collection. Subjects receive protein beverages in a non-transparent shaker.