We thank Witard and Mettler (2024) for their kind words regarding our recent work titled “The Anabolic Response to Protein Ingestion During Recovery From Exercise Has No Upper Limit in Magnitude and Duration In Vivo in Humans.” Our findings challenge several previous beliefs in the field that are central to support the concept of protein intake distribution: several moderately sized meals spread over the day may result in greater muscle anabolism compared with the same amount of protein consumed in less frequent but bigger meals or a more skewed intake pattern. Witard and Mettler share their perspective on our recent data and highlight some related literature to stimulate discussion regarding potential practical implications of our work. Here, we will briefly respond to their main discussion points.
Can the Anabolic Response to a Single Meal Inform About Protein Distribution?
We agree with Witard and Mettler that our study was not designed to investigate the impact of protein intake distribution on muscle protein anabolism per se. However, the belief that the anabolic response to protein ingestion is saturable and short-lived forms the main rationale/argument for the concept of protein intake distribution (Hudson et al., 2020; Layman, 2024; Lees et al., 2021; Murphy et al., 2016; Schoenfeld & Aragon, 2018). This concept is largely based on dose–response studies evaluating the anabolic response to the ingestion of a single meal. Our recent work applied a similar single-meal dose–response design to demonstrate that the anabolic response to the ingestion of a large(r) amount of protein is much greater than previously assumed when it is evaluated over a prolonged assessment period. Thus, our recent work challenges one of the central arguments that supports the protein intake distribution concept.
Much work has investigated the impact of protein intake distribution on muscle protein synthesis or muscle mass. These studies typically compare the impact of meal frequency (e.g., 40 g protein consumed every 6 hr vs. 20 g protein consumed every 3 hr), the impact of distribution pattern within the same meal frequency (e.g., three even-balanced meals vs. the majority of protein consumed in one of the main meals to resemble a Western diet), or time-restricted feeding (e.g., all food is consumed within an 8-hr period). In contrast to the protein intake distribution concept, the majority of such work has observed no impact on muscle protein synthesis or muscle mass (Hudson et al., 2020). This is despite investigations and meta-analysis in time-restricted feeding (Gu et al., 2022; Tinsley et al., 2019; Trepanowski et al., 2017), which represents an extreme model of (supposedly) suboptimal protein intake distribution where a large effect size would be expected. Therefore, we belief our recent work realigns single-meal dose–response studies with the overall findings of protein intake distribution studies that assessed either muscle protein synthesis rates or changes in muscle mass as clinical endpoints.
Efficiency of Dietary Protein-Derived Amino Acid Incorporation Into Skeletal Muscle Protein
We observed that ∼17% and ∼13% of the ingested protein-derived amino acids were incorporated into skeletal muscle protein in the 25 and 100 g protein intake treatments, respectively. Witard and Mettler argue that the higher percentage in the 25 g protein treatment would support a higher efficiency of (more frequent) moderate meals over (less frequent) larger meals. This argument is, however, incorrect as it overlooks one of the key findings from our work. Namely, the cumulative incorporation of dietary protein-derived amino acids into skeletal muscle protein was clearly saturated in the 25 g protein treatment but was still strongly increasing at the end of the 12-hr assessment period in 100 g protein treatment (see Figure 6C in Trommelen, van Lieshout, Nyakayiru, et al., 2023). We emphasized the importance of this finding since the total metabolic effect of an intervention will be underestimated over an assessment period that is insufficient in duration. It is clear from our work that the incorporation of dietary protein-derived amino acids becomes more similar between the 25 and 100 g treatments when considered over an even longer assessment period. Therefore, it would be inappropriate to compare these treatments in terms of efficiency as one of them has reached its maximal value, while the other was still rising at the end of the assessment period.
Extrapolation to Muscle Mass Gain
Urea Production
Witard and Mettler indicate that we did not assess urea production as a metabolic fate of amino acids. We assessed amino acid oxidation to investigate the previous belief that most of the protein ingested in amounts exceeding 20 g are oxidized. Following amino acid catabolism, nitrogen is eliminated through urea production. Therefore, urea production would represent an alternative measure of amino acid catabolism, a factor that we already accounted for by measuring amino acid oxidation.
Does Protein Distribution Have Beneficial Effects Beyond Protein Anabolism?
Witard and Mettler suggest that a more even protein intake distribution may be beneficial for other outcomes beyond muscle protein anabolism. While this is certainly possible, there are also many arguments that more frequent feeding can be detrimental to health. For example, in the longevity field, there is much interest in time-restricted feeding to improve health outcomes (e.g., cardiometabolic outcomes, cancer outcomes, or increase lifespan; Lee & Longo, 2011; Longo & Panda, 2016; Patikorn et al., 2021). The potential beneficial and/or detrimental effects of various protein distribution strategies remain debated but are currently not supported by sufficient high-quality evidence. However, our recent data imply that total daily protein intake may be more important than meal frequency as the main determinant of postprandial hyperaminoacidemia and its subsequent metabolic consequences (Figure 1).
—Schematic representation of plasma amino acid concentrations following different feeding strategies. It has been assumed that meal frequency determines the ratio between the fasted and fed state (A); however, total daily protein intake is likely a more important determinant (B). It has been assumed that regular meal moment throughout the date result in large amino acid fluctuations (C); however, this is based on research with free amino acids and rapidly digesting protein powders. In practice, athletes consume high-protein diets consisting out of whole foods in more complex, mixed meals which result in sustained hyperaminoacidemia with only relatively minor fluctuations based on meal frequency (D).
Citation: International Journal of Sport Nutrition and Exercise Metabolism 34, 5; 10.1123/ijsnem.2024-0107
Future Directions
Witard and Mettler discuss work by Areta et al. (2013) and Mallinson et al. (2023) as data to support the concept of protein intake distribution and how our recent work may not translate to trained females, respectively. For brevity, we cannot provide a more detailed discussion of the strengths and limitations of the referred work and how they relate to our recent work. Instead, we will provide some general recommendations for tracer studies investigating the concept of protein intake distribution:
- •Whole-Food Mixed Meals. Most studies assessing muscle protein synthesis rates have been performed with isolated amino acids or protein concentrates or isolates that are rapidly digested and/or absorbed, resulting in large fluctuations in plasma amino acid concentration throughout the day. In practice, athletes consume the majority of protein via whole-food mixed meals (Gillen et al., 2017). Such a diet will result in prolonged and moderate hyperaminoacidemia with relatively minor fluctuations (Justesen et al., 2022).
- •Postprandial Assessment Period. To properly assess postprandial protein handling, the assessment period in stable isotope tracer studies should be of sufficient duration to allow all of the ingested protein to reach its metabolic fate (protein synthesis or oxidation) (Trommelen, van Lieshout, Nyakayiru, et al., 2023).
- •Tracer Steady State. Different protein dose or protein intake patterns can largely disturb tracer steady-state conditions. To overcome these issues, intrinsically labeled protein can be applied in single meal and protein distribution studies to maintain postprandial tracer steady-state conditions (Justesen et al., 2022; Trommelen et al., 2021; Trommelen, van Lieshout, Nyakayiru, et al., 2023).
Practical Recommendations for Athletes
There is currently insufficient evidence to recommend a specific protein distribution pattern to athletes to optimize muscle anabolism. In practice, athletes consume 1.5–2.0 g protein per kilogram body mass per day, with the majority consumed as whole foods in mixed meals during breakfast, lunch, and dinner (Gillen et al., 2017). Such a diet likely results in continuous hyperaminoacidemia with relatively little fluctuations irrespective of the distribution pattern. In practice, this implies that athletes can focus on achieving their target total daily protein intake and can be more flexible with meal frequency than previously believed. The latter is an important message, as we are often asked by athletes if they should get up in the middle of the night to consume a bolus of protein to “optimize protein distribution and, as such, improve postexercise muscle conditioning.” We strongly recommend against this as the benefits of protein intake distribution are far from established and ingestion of a single 40 g bolus of protein in the evening will provide ample amino acids throughout the night without the need to sacrifice sleep quality (Res et al., 2012; Trommelen, van Lieshout, Pabla, et al., 2023).
We expect that some athletes and coaches would like protein distribution recommendations “just in case” of potential upside. Based on the current intake pattern of athletes, the addition of a presleep protein meal would be the best strategy to improve overall protein distribution. For per-meal protein recommendations, the ingestion of ∼20 g gives a strong stimulation of muscle protein synthesis over a 4-hr period. The ingestion of ≥40 g protein will further increase muscle protein synthesis by 10%–20% over that 4-hr period in healthy, young adults (Churchward-Venne et al., 2020; Macnaughton et al., 2016; Moore et al., 2009; Trommelen, van Lieshout, Nyakayiru, et al., 2023; Witard et al., 2014). When the next meal is not consumed within the next 4–5 hr, a protein dose of ≥40 g is recommended to maximize muscle anabolism over a more prolonged period. This is not only based on our recent work (Trommelen, van Lieshout, Nyakayiru, et al., 2023) but also supported by our extensive work on presleep feeding in which a dose of ≥40 g protein was shown to be required to observe a postprandial muscle protein synthetic response over a prolonged 7.5 overnight period (Kouw et al., 2017; Res et al., 2012; Snijders et al., 2019; Trommelen et al., 2018; Trommelen, van Lieshout, Pabla, et al., 2023).
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