by Eric Trexler, Ph.D.

We talk about protein pretty often around here, and for good reason. For starters, protein intake is one of the most important dietary variables we can modify to impact long-term development of strength and muscle mass. But that’s not the only reason for intrigue. There’s some very important protein research happening these days, and some of it is causing us to reconsider some long-standing beliefs. One example pertains to plant-based proteins and the broader concept of protein quality. While plant-based proteins have long been considered inferior to animal-based proteins, previous MASS articles (one, two) covered new research that casts doubt on this oversimplification. That’s great for determining which protein sources to utilize, but it doesn’t tell us much about one of the most common protein questions in the fitness world: how much protein can I actually “use” in a single meal?

This question has evolved over time. Several years ago, people typically asked how much protein they could “absorb” in a single meal. By now, a large portion of the fitness world has come to accept that absorption is rarely an issue or a limiting factor when it comes to dietary protein intake. Nowadays the most common iteration of the questions pertains to how much protein is needed to maximize the anabolic impact of a meal – in other words, how do I maximize the muscle-building effect of a meal without “wasting” protein. It’s often said that a mere 20-25 grams of protein should maximize the muscle protein synthesis response to protein ingestion, and that muscle protein synthesis can only be stimulated by protein ingestion every few hours (give or take). That seems like enough information to answer the underlying question, but the evidence is unsatisfactory when you start to think through the practical ramifications. For instance, if 20 grams of protein every few hours is the best we can do for building muscle, why shouldn’t a massive bodybuilder be able to maximize muscle growth with only 80-100 grams per day of protein?

As it turns out, the pre-existing research examining muscle protein synthesis in response to varying protein dosages has some pretty important limitations. Most notably, those studies tend to focus on fairly narrow ranges of protein intakes, tend to implement study conditions that maximize the rate of protein absorption (i.e., consuming whey protein in a fasted state), tend to examine responses over relatively short timescales (usually about six hours or so), and generally don’t involve full-body resistance training. The presently reviewed study by Trommelen et al (1) circumvents all three of these shortcomings, evaluating 12 hours of protein metabolism following the ingestion of 0, 25, or 100g of milk protein after a full-body resistance training workout. The results of this study caused quite a stir in the evidence-based fitness world, so let’s take a look at what they did and what they found.

Purpose and Hypotheses

Purpose

The purpose of the presently reviewed study was to assess “the time resolution of postprandial protein handling in response to the ingestion of moderate and large amounts of protein (0, 25, and 100 g) following exercise.”

Hypotheses

The researchers hypothesized that they would observe “a dose-dependent increase in the magnitude and duration of protein-derived amino acid availability and muscle and whole-body protein synthesis rates, with a negligible impact on amino acid oxidation.”

Subjects and Methods

The presently reviewed study included 36 healthy, recreationally active men between the ages of 18 and 40. To be included in the study, participants needed to be a generally healthy male, have a BMI between 18.5 and 30, and exercise 1-3 times per week. Subjects could not participate if they smoked, were lactose intolerant, had medical conditions that might interfere with study outcomes, or took medications that might interfere with study outcomes. After enrolling, participants were randomly assigned to consume 0g (control), 25g (moderate protein), or 100g (high protein) of intrinsically labeled milk protein after completing a single bout of exercise. The exercise bout was a whole-body resistance training session that lasted 60 minutes, which occurred immediately prior to beverage ingestion. Participants did 4 sets of each of the exercises (leg press, leg extension, lat pulldown, and chest press). Each exercise began with a set of 10 repetitions using 65% of 1RM, then 2-4 sets were performed with 80% of 1RM until volitional failure.

The researchers were interested in examining a wide range of outcomes related to the metabolic fate of the ingested protein. To make this possible, the protein in the beverages contained labeled versions of phenylalanine and leucine, and the researchers delivered intravenous infusions of labeled phenylalanine, tyrosine, and leucine. The researchers took blood samples and vastus lateralis muscle biopsies repeatedly over a 720-minute (12-hour) period, which allowed them to trace the protein’s fate and quantify various calculations related to protein metabolism. The researchers reported a tremendously large amount of data in their results, so I’ll simplify my reporting and restrict my focus to the most fitness-relevant outcomes.

Findings

In terms of whole-body protein metabolism, the researchers found that protein synthesis was significantly higher in the 100g protein condition than the 0g and 25g conditions. Protein breakdown was not significantly different among the three protein intake levels. Protein oxidation was significantly higher in the 100g protein condition than the 0g and 25g conditions, but the total magnitude of protein oxidation was small. As a result, net protein balance reflected a dose-response pattern; net balance was significantly higher in the 100g condition than in the 25g condition, which was significantly higher than in the 0g condition. These values are presented in Figure 1.

The fact that protein oxidation was highest in the 100g protein condition might lead some to assume that a meaningful amount of protein was “wasted.” However, this wasn’t the case. Protein oxidation was weakly correlated with relative protein dose (expressed as grams of protein per kilogram of body mass; R = 0.2689, p < 0.001), but the slope of this relationship was very shallow. So it’s true that relative protein dose was positively correlated with protein oxidation, but the overall magnitude of protein oxidation was pretty negligible. The relationship between relative protein dose and protein oxidation is presented in Figure 2. 

Given the dose-response elevation of protein synthesis, negligible impact of protein breakdown, and negligible impact of protein oxidation, net protein balance in the 100g protein condition was significantly greater than the 25g and 0g conditions. When expressed in relative terms (g/kg), protein dose explained 96% of the variation in net protein balance (p < 0.001). The relationship between relative protein dose and net protein balance is presented in Figure 3. 

As noted previously, this study reported a ton of data. That’s fantastic, but difficult to concisely relay in a MASS review. While the results presented above pertain to whole-body protein balance, you may rightfully be more interested in muscle-specific outcomes. Fortunately, muscle-level outcomes generally mirrored the dose-response relationships observed for whole-body protein synthesis and protein balance. As the researchers put it: “we observed a clear dose-dependent pattern in muscle protein synthesis rates when assessed over the entire 12-h postprandial period (100PRO > 25PRO > 0PRO).” In line with their hypothesis, the main advantage for the very high (100g) protein dosage was observed later in the postprandial period. Protein synthesis was 20% higher (100g versus 25g) from hours 0-4 after ingestion, but 40% higher from hours 4-12. While whole-body protein synthesis rates fell back to baseline at around 360 minutes after ingesting 25g of protein, rates remained elevated for around 720 minutes when 100g of protein was ingested. In other words, a very high protein dose appears to extend the time window of the anabolic response, thus leading to a larger cumulative impact on protein accretion when compared to a moderate dose.

The last finding I want to highlight pertains to “proxy measurements.” I’ve said time and time again that muscle protein synthesis is informative, but isn’t perfectly indicative of long-term muscle growth. Extending this specific claim to a more generalized heuristic, we shouldn’t assume that mechanistic measures are effective proxies for the outcomes we intend to make inferences about (unless we’ve got hard data to verify the assumption). In this study, the researchers looked at phosphorylation of proteins and expression of genes that are associated with anabolic processes in muscle. I won’t get too deep into the details here, but if you’re a muscle physiology aficionado, we’re talking about mTOR, p70S6K, myostatin, and so on. Despite observing differences related to whole-body and muscle-specific measures of protein balance and synthesis, the researchers “did not observe any impact of protein feeding on muscle protein signaling or muscle gene expression.” While these molecular responses tend to be short-lived, the anabolic response to protein feeding can be much longer, particularly when large doses of protein are consumed. So, we should heed the warning of the researchers when interpreting studies in the future: “the magnitude and/or time course of muscle molecular responses should not be used as a proxy to evaluate the magnitude of the postprandial anabolic response to feeding.”

Interpretation

If you want to do this “evidence-based fitness” thing the right way, consistency is the golden rule. You can’t cherry-pick evidence, and you can’t disregard a certain type of research when it contradicts your beliefs while embracing the same type of research when it supports your beliefs. In a previous MASS article about muscle protein synthesis studies, I stated the following:

“If you scour the literature, you’ll find several examples of isolated findings that lead to dubious conclusions if we assume that acute muscle protein synthesis is reliably predictive of long-term hypertrophy outcomes. So, in order to retain a somewhat cohesive and internally consistent understanding of the universe, I’ve simply let go of that assumption in many contexts.”

In that article, I discuss the many shortcomings of the literature assessing short-term muscle protein synthesis changes in response to varying types and doses of protein (2). My main argument was that this literature often leads to untenable conclusions when you assume that 1) these study protocols reflect common dietary practices and 2) muscle protein synthesis outcomes are reliably predictive of long-term body composition changes. As I further explained, “you can find studies suggesting that muscle protein synthesis can be maximally stimulated by only 20g of protein in young and healthy individuals (3), that muscle protein breakdown is of minimal importance in studies evaluating anabolic properties of various diet and exercise interventions (4), and that refractory periods dictate that we should only be able to maximally stimulate muscle protein synthesis around 4-5 times per day (5). If we jam these puzzle pieces together and take each of them at face value, it would support a virtually universal protein recommendation of 80-100g/day to maximize hypertrophy, but that’s incompatible with the longitudinal evidence that actually measures hypertrophy over time.”

To be clear, the presently reviewed study isn’t “just another muscle protein synthesis study” – this was a very rigorous study that circumvented or attenuated many of the methodological limitations that impact the typical protein synthesis study on this topic. Most notably, the researchers recruited previously trained participants, explored a large range of protein dosages, included a whole-body resistance training session, and measured outcomes over a longer time frame than the norm. Nonetheless, if you’ve previously unburdened yourself of the responsibility to make sense of the discordance between acute muscle protein synthesis studies and long-term hypertrophy outcomes, you can’t turn around and suggest that an acute muscle protein synthesis study definitely confirms your views on protein intake. So I won’t.

Instead, I’ll make a much more modest claim: this paper provides mechanistic evidence to help us better understand the disconnect between prior protein synthesis studies and longitudinal hypertrophy studies. For the sake of argument, let’s look at the common cluster of beliefs that muscle protein synthesis is maximally stimulated by only 20g of protein, that muscle protein breakdown is of minimal importance for hypertrophy, and that refractory periods dictate that we should only be able to maximally stimulate muscle protein synthesis every few hours or so. Many studies comparing the effects of high- versus low-protein diets involve a dietary pattern with roughly three meals per day. In such a scenario, 60g/day of protein should be just as effective as 200g/day of protein for supporting hypertrophy. If true, this would totally undercut and obscure the advantage of higher-protein diets, and meal frequency would be a very important predictor of hypertrophy. However, this doesn’t align with longitudinal hypertrophy research. In fact, the opposite is true; studies tend to consistently observe positive linear relationships between protein intake and hypertrophy outcomes across a broad range of intakes, with the relationship eventually plateauing at very high protein intake levels (6). In contrast, the impact of protein distribution is less clear and hints at a far less substantial impact than total protein quantity (7).

The previously reviewed study by Taguchi and colleagues (8) is a great example. The researchers compared the effects of higher (6 meals/day) and lower (3 meals/day) meal frequencies on body composition and appetite using a crossover design in competitive male rowers. Both diet interventions contained the same amount of total daily protein and calories and were consumed during a period of intentional weight gain. If 20g of protein every few hours is the best we can do with our protein intake, we would expect a clear advantage in favor of the higher-frequency condition. The math is pretty simple: six opportunities to maximally spike protein synthesis versus three. In reality, both diet conditions led to similar increases in body weight, body-fat percentage, fat mass, and fat-free mass (Table 1).

It might sound like I’m suggesting that protein distribution has absolutely no impact on hypertrophy outcomes, but I’m not quite ready to go that far. For example, a previous MASS article reviewed a study on “OMAD,” or the dietary practice of eating one meal per day (9). It was a crossover trial, so participants served as their own controls. While participants lost more lean mass during the OMAD condition (compared to three meals per day), the difference was small (-0.7kg versus -0.3kg) and was not statistically significant. I normally wouldn’t pay much attention to a mere 0.4kg difference (especially in a study without a resistance training component), but each diet condition lasted only 11 days – there was virtually no chance of observing large body composition changes, so attempting to extrapolate from tiny changes is the best we can do. Ultimately the diets were only 11 days long and only implemented by 11 participants, so this particular study constitutes very weak evidence if we’re interested in long-term hypertrophy outcomes.

A different MASS article reviewed a more generalizable study by Yasuda et al that evaluated the effects of two different diets (one with a high-protein breakfast and even protein distribution; another with a low-protein breakfast and uneven protein distribution) on strength and lean mass following 12 weeks of resistance training (10). The low-protein breakfast group only had two large daily protein doses, while the high-protein breakfast group had three. The study recruited 33 healthy men between the ages of 18-26 and measured strength and body composition outcomes before and after the diet intervention. Both study groups made gains in strength and lean mass over the 12-week study. The high-protein breakfast group had slightly larger increases for all five strength outcomes and total lean soft tissue, but none of these between-group differences were statistically significant. Further, the groups had virtually identical changes in appendicular (limb) lean soft tissue (1.14 ± 0.18 kg vs. 1.14 ± 0.17 kg), which means the observed differences in total lean soft tissue were almost exclusively attributable to differences in the trunk rather than the arms or legs. These results are presented in Figure 4.

There are a few important points to be made from these previous studies comparing one, two, three, and six protein doses per day. First, the benefits of higher protein frequency seem to diminish pretty quickly; if six meals per day fails to outperform three meals per day, I’m inclined to suggest that a mere three meals per day can effectively maximize hypertrophy outcomes (assuming you’re doing all the other important stuff). Second, even when we look at the low-frequency meal range that appears to be “suboptimal,” we’re talking about very small differences. For the Yasuda study comparing two large protein doses versus three (10), the vast majority of comparisons yielded non-significant differences. The same is true for the OMAD study discussed previously (9). 

Based on the evidence we have, I’m inclined to conclude that two protein doses per day is a bit better than one, three is slightly better than two, and you don’t have much to gain from going beyond three (unless it suits your preferences). However, the “disadvantage” of eating only 1-2 protein servings per day appears to be quite small, and I am absolutely certain that the vast majority of lifters can make fantastic (but slightly less than optimal) progress with this type of meal pattern. The presently reviewed study helps us better understand how a meal pattern with low-frequency protein distribution would be able to support hypertrophy surprisingly well in the more applied studies published to date. Nonetheless, I want to reiterate that my conclusions on this topic are tentative in nature; when you consider how frequently health professionals and fitness enthusiasts discuss and debate protein distribution, the lack of applied research is glaring. We’ll need more longitudinal studies that directly assess hypertrophy outcomes if we want to draw conclusions with a high level of confidence.

Next Steps

I’d like to see a pretty straightforward independent replication of this study. Not because I distrust the results or the lab they came from, but because independent replication is a foundational tenet of rigorous science. I’m particularly interested in resolving a mechanistic question that relates to the role of protein synthesis versus protein breakdown. The present study isn’t the first to suggest that the anabolic response to a meal has “no practical limit” with regards to protein dosage. However, a high-profile review paper making this argument in 2018 largely focused on how very high protein doses can suppress muscle protein breakdown and increase protein synthesis in non-muscle tissues (6). The presently reviewed study instead emphasizes a prolonged duration of muscle protein synthesis, so I’d like to get additional clarification on that. In addition, we simply need more studies that directly measure what we care about: how do different protein distributions actually impact hypertrophy over the course of at least 3 months of structured resistance training? This question is remarkably understudied despite its practical utility.

Application and Takeaways

Fitness enthusiasts have long suggested that you should eat around 20-30 grams of protein in several equal increments throughout the day if you wish to optimize protein synthesis and muscle growth. Mounting evidence suggests that we can make use of substantially larger protein doses and that we have a lot more wiggle room when it comes to protein distribution. While I still (tentatively) contend that 3-6 protein servings per day are likely to optimize long-term hypertrophy, evidence suggests that the difference between 2/day and 3/day is pretty slim, and I am absolutely certain that you can make terrific (but probably not maximal) progress with only one meal per day.

References

1. Trommelen J, van Lieshout GAA, Nyakayiru J, Holwerda AM, Smeets JSJ, Hendriks FK, et al. The anabolic response to protein ingestion during recovery from exercise has no upper limit in magnitude and duration in vivo in humans. Cell Rep Med. 2023 Dec 19;4(12):101324.
2. Witard OC, Bannock L, Tipton KD. Making Sense of Muscle Protein Synthesis: A Focus on Muscle Growth During Resistance Training. Int J Sport Nutr Exerc Metab. 2022 Jan 1;32(1):49–61.
3. Witard OC, Jackman SR, Breen L, Smith K, Selby A, Tipton KD. Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise. Am J Clin Nutr. 2014 Jan;99(1):86–95.
4. Trommelen J, Betz MW, van Loon LJC. The Muscle Protein Synthetic Response to Meal Ingestion Following Resistance-Type Exercise. Sports Med. 2019 Feb 1;49(2):185–97.
5. Mitchell WK, Phillips BE, Hill I, Greenhaff P, Lund JN, Williams JP, et al. Human skeletal muscle is refractory to the anabolic effects of leucine during the postprandial muscle-full period in older men. Clin Sci. 2017 Oct 27;131(21):2643–53.
6. Kim IY, Deutz NEP, Wolfe RR. Update on maximal anabolic response to dietary protein. Clin Nutr. 2018 Apr;37(2):411–8.
7. Hudson JL, Iii REB, Campbell WW. Protein Distribution and Muscle-Related Outcomes: Does the Evidence Support the Concept? Nutrients. 2020 May 16;12(5):1441.
8. Taguchi M, Hara A, Murata H, Torii S, Sako T. Increasing Meal Frequency in Isoenergetic Conditions Does Not Affect Body Composition Change and Appetite During Weight Gain in Japanese Athletes. Int J Sport Nutr Exerc Metab. 2021 Mar 1;31(2):109–14.
9. Meessen ECE, Andresen H, van Barneveld T, van Riel A, Johansen EI, Kolnes AJ, et al. Differential Effects of One Meal per Day in the Evening on Metabolic Health and Physical Performance in Lean Individuals. Front Physiol. 2022 Jan 11;12:771944.
10. Yasuda J, Tomita T, Arimitsu T, Fujita S. Evenly Distributed Protein Intake over 3 Meals Augments Resistance Exercise-Induced Muscle Hypertrophy in Healthy Young Men. J Nutr. 2020 Jul 1;150(7):1845–51.