The notion that the time of day, frequency, and distribution of daily dietary protein intake could influence protein metabolism dates back to the late 1930s. Researchers at the time manipulated dietary protein consumption frequency and distribution to effect (1) protein/amino acid balance and (2) nitrogen balance. These two balances are thought to be connected through the free amino acid pool. Constituting only a fraction of the total nitrogen pool, the free amino acid pool is maintained within narrow limits; therefore, when the body is in nitrogen balance (input = output), then we presume protein synthesis and breakdown are in balance [
2]. However, nitrogen balance studies have their limitations [
55,
56,
57,
58,
59,
60]. With the advent of stable isotope amino acid methodologies, we can more accurately track the flux of amino acids in and out of tissues. Accordingly, the flux of amino acids from skeletal muscle and their incorporation into muscular proteins were emphasized in the 2000s to quantify protein synthetic rates in humans. Evidence from acute stable isotope studies provided cursory support for the within-day day protein distribution to impact skeletal muscle mass and function [
34,
35,
36]. This prompted more research using prospective randomized controlled trial designs in which the relationship between protein distribution and skeletal muscle-related outcomes beyond acute changes in MPS could be further elucidated. This section will provide an in-depth analysis of the more valid nitrogen balance studies and of the more recent longitudinal randomized controlled trials on whole-body composition outcomes.
3.2.1. Nitrogen Balance
For most the 20th century, highly controlled nitrogen balance studies were considered the gold standard for studying protein metabolism; in fact, results from nitrogen balance studies still inform adult protein requirements [
1]. Although recent protein metabolism research has emphasized the protein distribution concept, it was of scientific interest as early as 1939. The first study, to our knowledge, was performed using a single male participant by Cuthbertson and Munro [
61]. Their initial research was focused on understanding the relationship between carbohydrate and protein metabolism; this inevitably led them to studying the effect of the within-day protein intake frequency—a form of protein distribution. Since their initial research, the effect of protein distribution on nitrogen balance has been studied at least 11 more times [
7,
38,
39,
61,
62,
63,
64,
65,
66,
67,
68,
69]. Many of these studies, however, do not sufficiently meet the criteria for a valid nitrogen balance experiment [
70] (e.g., appropriate stabilization periods; periods consuming the controlled diets must be long enough; corrections for integumental and miscellaneous losses; urine and fecal collections must be precisely timed and complete). Considering the evidence in light of these criteria, we considered only two of the 12 studies—one study shows an effect of protein distribution [
38] and the other shows that protein distribution does not influence nitrogen balance [
39,
62] (
Table 3).
Arnal et al. [
38] were the first to study protein distribution in older women. Participants were randomized to consume either 80% of their protein at lunch (unbalanced) or a more balanced protein distribution pattern (21:31:19:28% protein/meal). After 14 d, the unbalanced group had a more positive nitrogen balance and a maintenance of fat-free mass; fat-free mass slightly decreased (~−0.3 kg) in the balanced group. This was attributed to the greater 24 h whole-body protein synthesis in the unbalanced group than the balanced group, although fed-state protein synthesis rates were not different. The same study design was replicated in younger women by the same group [
39]. However, this time the authors reported no difference between groups in whole-body fat-free mass, protein turnover, and protein synthesis and breakdown. While there was no statistically supported difference in whole-body nitrogen balance, nitrogen retention was 1.5 times higher with the balanced vs unbalanced distribution (
p = 0.16). The authors highlight the difference in nitrogen balance between groups was 23 mg N·kg fat-free mass
−1·d
−1 (balanced > unbalanced); this was similar in magnitude to the difference reported in the older females (27 mg N·kg fat-free mass
−1·d
−1; balanced < unbalanced) [
38,
39].
These results from Arnal et al. [
38,
39] are consistent with an anabolic resistance to feeding commonly observed among older adults. Although four balanced meals were consumed, the quantity of protein at each meal may have been too low to maximally, or at least meaningfully, stimulate MPS [
38], i.e., they consumed a balanced but not optimal protein distribution. Conversely, while consuming the unbalanced protein distribution, participants likely maximized MPS after the lunch meal. Even if MPS after lunch was not maximized, these results that suggest aggregate protein synthesis was higher after consuming an unbalanced distribution pattern that likely maximized MPS at least once. Among younger adults who are more “sensitive” to dietary protein/amino acid intake, the lower protein doses consumed in a balanced protein distribution likely triggered a more robust anabolic response to feeding [
39]. The postprandial MPS responses while consuming the unbalanced distribution likely were not greatly affected; the maximum achievable MPS rate is not different between younger and older adults [
71]; therefore, the MPS responses between groups after lunch were likely comparable.
3.2.2. Body Composition
Acute human clinical studies often provide the foundational science used to design longitudinal studies. Longitudinal randomized controlled trials have the benefit of inherently capturing daily fluctuations in hormonal concentrations, energy intakes, and exposure to stress; these factors can have profound impacts on both phenotypical and functional outcomes. Chronic feeding studies are necessary to assess the applicability and efficacy of acute studies in real-world settings. Only five longitudinal studies to date (to our knowledge) have investigated the effect of protein distribution on body composition [
43,
62,
72,
73,
74]. One study supports consuming a balanced protein distribution [
73], three report null results [
43,
62,
74], and one supports consuming an unbalanced distribution for lean mass gains [
72] (
Table 4).
Among all randomized controlled trials, only one study supports consuming a balanced protein distribution to support lean mass gains [
73]. In this study, Yasuda et al. [
73] tested the effect of providing participants with either a low or high-protein breakfast. The high-protein breakfast was achieved by the addition of a protein supplement; the same supplement was consumed at dinner by the low-protein breakfast group. Both groups experienced resistance-training induced increases in lean body mass over time; however, the increases tended to be greater in the high-protein breakfast group (2.5 ± 0.3 kg vs. 1.8 ± 0.3 kg;
p = 0.056); there was no effect on appendicular lean mass changes. Notably, the balanced group (high-protein breakfast) had a lower total protein intake (1.3 g·kg
−1·d
−1) than the unbalanced group (lower-protein breakfast; g·kg
−1·d
−1). However, due to small samples sizes in both groups, albeit a large effect size (
d = 0.7), this study is likely more proof of concept. Only one other study [
74] seemingly provided enough protein per meal to be considered an “optimal” distribution pattern; however, contrary to Yasuda et al. [
73], Hudson et al. [
74] did not report an effect of protein distribution on lean mass changes. Based on the research by Mamerow et al. [
34], Hudson et al. [
74] prescribed 30 g of protein per meal (~0.3 g·kg
−1·meal
−1); this should have provided a “safety margin” of ~0.1 g·kg
−1 [
10]. As mentioned in the acute protein ingestion section above, their estimates are based on studies utilizing isolated intact proteins [
10]. Mixed-nutrient meals, analogous to the one meal by Hudson et al. [
74], contain multiple whole-food protein sources; the variability in protein quality and the difference in food matrices alters the protein digestion and amino acid absorption kinetics compared with protein supplements [
49]. Underscoring this point, Kim et al. [
43] found that peak plasma leucine concentrations after consuming a whole-food meal were less than 50% of the peak concentration achieved from consuming an essential amino acids mixture containing twice the leucine quantity. Compared to a supplement, the protein quantity within mixed-nutrient meals likely needs to be greater. Yasuda et al. [
73] prescribed a protein supplement, in addition to the standardized whole-food breakfast, to attain the higher-protein meal. Sufficient quantity (0.33 g·kg
−1) and quality (whole foods and supplement) may have been adequate to achieve a saturable dose and promote an optimal distribution. Although Hudson et al. [
74] prescribed a balanced protein distribution, they may not have prescribed an “optimal” protein distribution due to the quality of protein consumed in the mixed meals. Conversely, the unbalanced protein distribution may have provided the protein dose required to stimulate MPS maximally after dinner; the result may have been comparable daily synthesis rates. The same reasoning could be used to explain the null results among the two other studies [
43,
62]: they prescribed balanced but not “optimal” protein distribution patterns. These arguments, however, are only conjecture; none of the studies measured postprandial MPS rates.
The research by Bouillanne et al. [
72] in hospitalized older adults showed that consuming an unbalanced protein distribution supported lean mass gains, while consuming a balanced protein distribution resulted in a loss of lean mass. Similarly, Hudson et al. [
74] found younger adults in energy restriction trended (
p = 0.067) towards losing less lean mass when they consumed an unbalanced protein distribution (−0.5 kg) versus consuming a balanced protein distribution (−1.5 kg). These results [
72,
74] seemingly agree with the observational results from Loenneke et al. [
27] and Loprinzi et al. [
28] and the nitrogen balance data provided by Arnal et al. [
38]: consuming ≥1 meal with sufficient protein to theoretically maximize MPS may be better for lean body mass retention than consuming three balanced meals with “insufficient” protein to be considered optimal.
While protein quantity within meals may partially explain the null results, the interplay between the anticipated effect size and the studies durations could provide further context. Per annuum, lean mass decreases by approximately 1%–2% on average in adults who typically consume an unbalanced protein distribution [
75,
76]. We assume the “best-case scenario” for consuming a balanced protein distribution in adults not purposefully altering their body composition over the year would be a retention of lean mass. The amount of lean mass possibly retained over 12 months, being relatively small, is difficult to detect with current techniques. Measuring a differential change between groups who retained and who lost lean mass over 2–4 months, the length of the studies presented here, may be even less feasible. Aligning with this, Kim et al. [
43] hypothesized that consuming a balanced vs unbalanced protein distribution over 8 weeks would increase lean mass [
77]; however, they showed that lean mass did not differentially change. Even with weight loss interventions, where lean mass loss is expected, both Adechian et al. [
62] and Hudson et al. [
74] reported that lean mass did not differentially change between groups who were energy-restricted even though the interventions were longer. Bouillanne et al. [
72] reported differential lean mass changes after only 6 wk; however, they studied a hospitalized, malnourished population. Among a population experiencing lean mass loss above the 1%–2% rate, an effect of protein distribution may be measurable with our current techniques under “shorter” durations.
An exercise stressor has been proposed to provide the “optimal environment” for protein distribution to induce differential changes in lean mass, including skeletal muscle, over a few months. Compared to protein ingestion while being sedentary, resistance training “sensitizes” the muscle to promote greater MPS [
47]. Theoretically, consuming a balanced distribution of protein among daily meals would stimulate MPS more frequently, in turn enhancing resistance training-induced lean mass and muscle accretion [
44]. An alternative hypothesis is that manipulating dietary protein distribution concurrently with resistance training would not influence lean mass and muscle accretion because the anabolic effect of resistance training is superior to the relatively less robust effect of protein distribution [
78]. No study to date has directly tested these opposing hypotheses regarding body composition.
As discussed, postprandial MPS partly determines changes in the skeletal muscle protein pool; however, we must acknowledge that both acute and chronic studies have several limitations. First, skeletal muscle changes in response to repeated exposures to both catabolic and anabolic stressors. These stressors include, but are not limited to, hormonal concentrations, energy status, and exercise training status. Their influence on lean mass changes cannot be accurately predicted from acute studies because they fluctuate and persist beyond the durations measured in laboratory settings. Second, the saturable dose estimates were established by sampling a single muscle—the
vastus lateralis. While MPS does not vary by either anatomic location or fiber type [
79], lean mass is not solely composed of skeletal muscle. Changes in lean mass also reflect changes in organ tissues and water. Third, hydration status can have a particularly profound influence on lean mass because water fluctuates to a greater magnitude than the muscle protein pool. In an attempt to improve the congruence between lean mass and skeletal muscle, a four-compartment model of body composition was developed to factor-in fluctuation in total body water [
80,
81]. This would get us closer to measuring changes in the protein pool alone. Another viable alternative to estimating changes in the protein content of skeletal muscle is to measure appendicular lean mass. The appendicular regions have more congruence with skeletal muscle than whole-body lean mass for the following: it is devoid of organs, is less prone to major fluctuations in body water, and is comprised primarily of skeletal muscle. However, surrogate markers of the skeletal muscle protein pool should always be interpreted with caution.
3.2.3. Future Research Directions for Chronic Protein Ingestion Research
We argued that the lack of evidence in support of an optimal protein distribution may be explained by both inadequate protein quantity and quality within meals; this resulted in balanced but not optimal protein distributions. The saturable dose estimates we used to critique meal protein quantity are based on isolated intact proteins. However, importantly, the vast majority of protein consumed comes from protein-rich foods, not supplements. Establishing the relative saturable doses using whole-food animal- and plant-protein sources will facilitate conducting optimal distribution studies while participants consume varied protein-rich foods.
Recommendations in the scientific literature are made for older adults to consume 0.4–0.6 g·kg
−1·meal
−1 to promote muscle retention [
82]. This equates to 1.2–1.8 g·kg
−1·d
−1, which is 50% to 125% higher than the recommended dietary allowance and ~15% to 110% higher than the average daily protein intake amongst older adult men and women [
19]. Currently, there is no direct evidence from randomized controlled trials to support consuming 1.2–1.8 g·kg
−1·d
−1 in a balanced protein distribution to promote muscle size, strength or quality. There is evidence these quantities of protein intake do promote beneficial changes in lean mass; however, this is compared to consuming lower-protein diets [
83,
84,
85,
86]. In these studies, the higher protein intakes may have been achieved by pragmatically adding the additional protein to breakfast and lunch; the dinner meal may not have been a good candidate for additional protein because it is usually the largest protein-containing meal. Therefore, it is possible we have evidence that a balanced higher-protein diet is more beneficial for lean mass than consuming an unbalanced lower/normal protein diet. We currently lack sufficient evidence to determine the effect of protein distribution on lean mass when total protein intake is matched at either lower or higher protein intakes.
Arguably, research designed to measure muscular function outcomes may be more meaningful than those measuring lean mass. Skeletal muscle strength and function, rather than mass, is often associated with health and longevity among aging adults [
87]. Shifting our research focus away from measuring mass and towards assessing function may be the future of protein ingestion research.