Dietary protein and amino acid intakes for mitigating sarcopenia in humans

Abstract

Adult humans generally experience a 0.5–1%/year loss in whole-body skeletal muscle mass and a reduction of muscle strength by 1.5–5%/year beginning at the age of 50 years. This results in sarcopenia (aging-related progressive losses of skeletal muscle mass and strength) that affects 10–16% of adults aged ≥ 60 years worldwide. Concentrations of some amino acids (AAs) such as branched-chain AAs, arginine, glutamine, glycine, and serine are reduced in the plasma of older than young adults likely due to insufficient protein intake, reduced protein digestibility, and increased AA catabolism by the portal-drained viscera. Acute, short-term, or long-term administration of some of these AAs or a mixture of proteinogenic AAs can enhance blood flow to skeletal muscle, activate the mechanistic target of rapamycin cell signaling pathway for the initiation of muscle protein synthesis, and modulate the metabolic activity of the muscle. In addition, some AA metabolites such as taurine, β-alanine, carnosine, and creatine have similar physiological effects on improving muscle mass and function in older adults. Long-term adequate intakes of protein and the AA metabolites can aid in mitigating sarcopenia in elderly adults. Appropriate combinations of animal- and plant-sourced foods are most desirable to maintain proper dietary AA balance.

Keywords:

• amino acids
• diet
• health
• nutrition
• protein intake
• skeletal muscle

Introduction

Skeletal muscle is a major organ in humans, representing approximately 40% and 32% of the total body mass in 30-year-old healthy men and women, respectively (Janssen et al. 2000). The whole body and skeletal muscle of adults contain approximately 15% and 20% protein, respectively (Duda et al. 2019). Thus, the 70-kg man and the 60-kg women have 10.5 and 9.0 kg protein, respectively, and their skeletal muscle accounts for 53% and 43% of the whole-body protein, respectively. Notably, skeletal muscle mass, which depends on the balance between the rates of protein synthesis and degradation, is relatively constant between 18 and 45 years of age in both men and women and thereafter decline (Janssen et al. 2000).

Sarcopenia is an aging-related progressive loss of skeletal muscle mass and function (Cruz-Jentoft et al. 2019) and affects 10–16% of adults aged 60 years or older worldwide (Yuan and Larsson 2023). This health problem can start as early as 27 years of age in sedentary men and women (Silva et al. 2010). Without preventative methods, adults generally experience a 0.5–1% loss/year in the whole-body skeletal muscle mass (including a loss of leg muscle by 1–2% per year) and a reduction of leg muscle strength by 1.5–5% per year) from the age of 50 years, with the rate of loss accelerating from 70 years of age onwards (Cruz-Jentoft et al. 2019; Lonnie et al. 2018). Notably, in individuals with limited physical activity, there is a 30–50% decrease in skeletal muscle mass and strength between the ages of 30 and 80 years (Daley and Spinks 2000). Sarcopenia results in physical weakness, disability, dependent living, and a diminished quality of life, while increasing the risks of falls, fractures, morbidity, and mortality, as well as health care expenditure, costly hospitalization, and extended rehabilitation (Larsson et al. 2019). With an increase in life expectancy due to improvements in living conditions and health management, the global aging population is increasing. For example, it has been estimated that by the year 2050, 20% of the U.S. population will be aged > 65 years (Mather, Jacobsen, and Pollard 2015). This will be associated with a dramatic increase in the prevalence of sarcopenia, which will reduce the longevity of humans and the quality of life. Preserving skeletal muscle mass and function is crucial for elderly adults to remain healthy and functionally independent.

Rates of protein synthesis and degradation in skeletal muscle are affected by physiological, pathological, and nutritional factors, including muscle contractility, insulin sensitivity, and stress as well as dietary intakes of energy, protein, amino acids (AAs), lipids, carbohydrates, vitamins, and minerals (Suryawan and Davis 2011; Wu 2022). A loss of muscle protein results from a lower rate of its synthesis than the rate of its degradation. Much evidence shows that an insufficient intake of protein, especially animal-sourced protein, contributes to the development of sarcopenia in men and women (Lancha et al. 2017; Putra et al. 2021). A national trend of nutrition is toward a limitation of consumption of animal-sourced protein in the U.S. (Lau et al. 2023; USDA 2021). For example, annual beef consumption per capita by American adults aged 19–59 years had declined by 20.8% from 21.2 kg in 2005 to 16.8 kg in 2015 (Lau et al. 2023). This is due, in part, to an inadequate understanding of aging-related nutrition and metabolism of protein and AAs in humans. The objective of this review article is to fill in this critical gap of knowledge to beneficially mitigate sarcopenia in humans and improve their health and well-being.

Anabolic resistance of protein synthesis in skeletal muscle with aging

There are reports that basal rates of mixed or myofibrillar protein synthesis in skeletal muscle, which are measured in the overnight (or 12-h)-fasted state, either do not differ between young (a mean age of 20 to 28 years) and older (a mean age of 61 to 75 years) adults (Cuthbertson et al. 2005; Fry and Rasmussen 2011) or are 15–25% lower in older than in young adults (Welle et al. 1993; Yarasheski, Welle, and Nair 2002), depending on nutritional and physiological states. Short et al. (2004) reported that the basal rates of mixed protein synthesis in the skeletal muscle of untrained men and women aged 19 – 87 years declined with advancing age at 3.5% per decade. However, results of most, but not all, studies have revealed that the response of skeletal muscle to dietary protein intake or resistance (strength) exercise in terms of mixed or myofibrillar protein synthesis is diminished by 20–35% with aging (Dorrens and Rennie 2003; Farnfield et al. 2012; Nair 1995; Paddon‐Jones and Rasmussen 2009; Wall et al. 2015). This phenomenon is termed the anabolic resistance of skeletal muscle with aging, likely because of impaired mechanistic target of rapamycin (mTOR) cell signaling (Cuthbertson et al. 2005), as well as reductions in the availability of certain AAs in the plasma and the uptake of AAs (e.g., Phe) by skeletal muscle (Katsanos et al. 2005). Insulin insensitivity in elderly men and women (a mean age of 69 years) is positively associated with elevated plasma concentrations of methylarginines (Marliss et al. 2006), which are inhibitors of the synthesis of nitric oxide (NO, the major vasodilator) from L-arginine in vascular endothelial cells (Wu et al. 2021). Thus, due to anabolic resistance with aging, elderly adults (e.g., 71 years of age) require 140% more protein intake (0.60 vs 0.25 g/kg body weight [BW] in a meal) to stimulate muscle protein synthesis at the same rate as that in young adults (Moore et al. 2015). Nonetheless, older adults can respond to elevated dietary protein intake with increases in protein synthesis and protein accretion in their skeletal muscle. Bauer et al. (2013) recommended a daily intake of 1.0–1.2 g and ≥ 1.2 g protein/kg BW for older people (> 65 years of age) with minimum physical activity and regular exercise (endurance or resistance), respectively, to maintain or regain lean body mass and function.

Intake and digestion of dietary protein by adult humans

Based on food intake data from 11,680 adults [51–60 years of age (n = 4,016), 61–70 years of age (n = 3,854), and 71 years of age and older (n = 3,810)] from the 2005– 2014 National Health and Nutrition Examination Survey (NHANES), Krok-Schoen et al. (2019) reported that a large proportion of U.S. adults did not meet the current Institute of Medicine (IOM)-recommended protein intake of 0.8 g/kg BW/day. Specifically, across all age categories, dietary protein intakes were decreased with age, with up to 50% of the oldest adults not meeting the protein intake recommendation due, in part, to poor food quality (e.g., inadequate amounts of animal-sourced food; Krok-Schoen et al. 2019). The percentages of men consuming < 0.8 g protein/kg BW/day are as follows: 51–60-year-old, 31%; 61–70-year-old, 37%; and > 70-year-old, 42%, and the percentages of women consuming < 0.8 g protein/kg BW/day are as follows: 51–60-year-old, 45%; 61–70-year-old, 48%; and > 70-year-old, 50% (Krok-Schoen et al. 2019). Based on the protein intake of 1.0–1.2 g/kg BW/day currently proposed by nutrition experts, even more adults in the U.S. do not have sufficient protein intake for optimum health including skeletal muscle health (Krok-Schoen et al. 2019). Likewise, dietary protein intakes of 29%, 54%, and 76% of community-dwelling older adults in the European union are below 0.8, 1.0, and 1.2 g/kg BW/day, respectively (Hengeveld et al. 2020).

Proteases in the lumen of the stomach, as well as proteases and peptidases in the lumen of the small intestine and on the apical membrane of its enterocytes are responsible for the hydrolysis of dietary proteins into small (di- and tri-) peptides and free AAs (Moughan and Rutherfurd 2012; Wu 2022). Aged adults have a sufficient ability to digest dietary protein and absorb the small peptides and free AAs, but the catabolism of AAs (e.g., branched-chain AAs) by the splanchnic bed (the portal-drained viscera plus liver) is greater in older than young adults (Boirie, Gachon, and Beaufrère 1997). Thus, given the same amount of ingested protein, less dietary AAs are available for use by extra-intestinal organs (including skeletal muscle, heart, kidneys, and brain) in elderly than in young adults. In support of this notion, Gorissen et al. (2020) reported that 45% and 51% of phenylalanine in dietary protein entered the blood circulation in 71- and 22-year-old adults, respectively, during a 5-h postprandial period. This should be taken into consideration when recommending protein intakes for elderly adults to meet their requirements for AAs and prevent the development of sarcopenia during the aging process.

Concentrations of free AAs in the plasma (or serum) of young vs older adult humans

Because of insufficient protein intake and the reduced entry of dietary AAs into the blood circulation in elderly adults (see above), concentrations of AAs in their serum or plasma would be expected to be reduced, compared with young adults. This is indeed the case for most AAs, because the circulating levels of many AAs (including arginine, glycine, serine, branched-chain AAs, methionine, threonine, and tryptophan) are lower in elderly than young adults (Table 1). A decrease in the circulating level of arginine in older than young adults results, in part, from a lower rate of endogenous synthesis (Deutz et al. 2018), as reported for aged mice (Wu et al. 2022). Note that the circulating levels of AAs are regulated by multiple factors, including the dietary intake of AAs, intestinal and extraintestinal catabolism and synthesis of AAs, and whole-body synthesis and degradation of protein. Thus, elevations in plasma or serum concentrations of BCAAs in some older than in young adults (Caballero, Gleason, and Wurtman 1991; Menni et al. 2013) may not necessarily indicate an adequate intake of these AAs but rather may reflect a net increase in whole-body protein degradation in the former. To our knowledge, previous investigators did not determine 4-hydroxyproline (a major AA in the whole body) in the serum or plasma of humans. Interestingly, studies involving rodents (mice and rats) also indicated lower concentrations of some AAs (including arginine, glycine, 4-hydroxyproline, serine, branched-chain AAs, methionine, and tryptophan) in elderly than in younger animals (Supplemental Table 1) (Gross et al. 1991; Hartman et al. 1997; Houtkooper et al. 2011; Jeon et al. 2018; Seo et al. 2016; Suárez et al. 2016; Uchitomi et al. 2019; Varshavi et al. 2018; and Yeung and Friedman 1991). Thus, elderly mammals such as humans are likely deficient in some AAs [including so-called nutritionally nonessential AAs (NEAAs; i.e., the AAs that are synthesizable de novo)] that are crucial for optimum health including skeletal muscle health, increasing risk for sarcopenia during aging.

Table 1. Changes in the concentrations of amino acids (AAs) in the serum or plasma of young versus older adult humans.

AA	Serum					Plasma
	Kouchiwa et al. (2012)^a	Dunn et al. (2014)^b	Menni et al. (2013)^c	Yu et al. (2012)^d	Pitkänen et al. (2003)^e	Chak et al. (2019)^f	Takada et al. (2020)^g	Darst et al. (2019)^h	Katsanos et al. (2005)^i	Caballero et al. (1991)^j	Foroumandi et al. (2018)^k	Volpi et al. (2000)^l	Deutz et al. (2018)^m
Ala	↑ (♂; ♀)	–	–	–	–	–	↑ (+17)	–	–	NR	–	–	–
Arg	↑ (♂; ♀)	–	–	–	–	↓	–	–	–	NR	–	–	–
Asn	↑ (♂; ♀)	–	–	–	↓ (–16%)	–	–	–	–	NR	↑ (+62%)	–	–
Asp	↑ (♂)	–	↓	–	↓ (–42%)	–	–	–	–	NR	↓ (–27%)	↑ (+37%)	↑ (+42%)
Cit	↑ (♂; ♀)	–	↑	–	↑ (+24%)	–	↑ (+22%)	↑	NR	NR	↑ (+20%)	NR	↑ (+37%)
Cys	–	↓	–	–	↑ (+182%)	–	↑ (+67%)	–	–	NR	NR	NR	NR
Glu	↑ (♂; ♀)	–	↑	–	↓ (–26%)	–	↑ (+22%)	–	–	NR	–	NR	↑ (+75%)
Gln	↑ (♀)	–	↑	–	↓ (–13%)	–	–	↑	–	NR	NR	–	–
Gly	↓ (♂;)	–	–	–	–	–	–	–	↓ (–32%)	NR	↑ (+46%)	↓ (–30%)	–
His	↓ (♂;)	–	↓	↓	↓ (–20%)	–	–	↓	↓ (–22%)	NR	–	NR	–
Hyp	NR	NR	NR	NR	NR	NR	NR	NR	NR	NR	NR	NR	–
Ile	–	–	–	–	–	–	–	–	–	↑ (♀; +26%)	↓ (–31%)	–	–
Leu	↓ (♂;)	–	↑	–	↓ (–18%)	–	–	↓	–	↑ (♀; +22%)	↓ (–34%)	–	–
Lys	↑ (♀)	–	–	–	↓ (–7%)	–	–	–	–	NR	↑ (+38%)	–	_
Met	↓ (♂;)	↓	–	–	↓ (–13%)	–	–	–	–	–	–	–	–
Orn	↑ (♂; ♀)	–	↑	–	–	↑	↑ (+22%)	↑	NR	NR	–	NR	–
Phe	↑ (♂; ♀)	–	–	–	↓ (–13%)	–	↑ (+15%)	–	–	↑ (♀; +7%)	↑ (+29%)	–	–
Pro	↑ (♀)	–	–	–	NR	–	–	–	–	NR	NR	NR	–
Ser	↓ (♂; ♀)	↓	↓	–	↓ (–14%)	↑ (♀)	↓ (–15%)	↓	–	NR	–	↓ (–47%)	↓ (–17%)
Tau	↓ (♂)	–	–	–	↓ (–18%)	–	↑ (+36%)	–	NR	NR	NR	NR	↑ (+24%)
Thr	↓ (♂; ♀)	↓	–	–	↓ (–22%)	–	–	↓	–	NR	↓ (–23%)	↓ (–43%)	–
Trp	–	↓	↑	↓	↓ (–12%)	–	–	↓	–	↓ (♂; −14%)	NR	–	–
Tyr	↑ (♂; ♀)	↑	↑	–	–	↑ (♀)	↑ (+21%)	↑	–	↑ (♀; +14%)	NR	–	–
Val	–	–	–	–	↓ (–11%)	–	–	–	–	↑ (♀; +20%)	↓ (–30%)	–	–

In all these studies, blood samples were obtained from study participants after an overnight fast. The metabolomics studies of Dunn et al. (2014), Menni et al. (2013), Yu et al. (2012), Chak et al. (2019), and Darst et al. (2019) did not provide quantitative data on either serum/plasma amino acid concentrations or their percent changes in older versus younger adults.

^a Regression analysis of adults (men and women) aged ≤29 to ≥75 years. There were 5 to 51 men per age group and 7 to 40 women per age group. The percent (%) increases in older men (≥75 years of age) than in younger men (≤29 of age) are: Ala, 23; Arg, 12; Asn, 6.7; Cit, 27; Glu, 43; Orn, 18; and Tyr, 16; whereas the percent (%) decreases in older men (≥75 years of age) than in younger men (≤29 of age) are: Gly, 12; His, 5.2; Leu, 3.3; Met, 3.5; Ser, 3.0; Tau, 2.8; and Thr, 5.7. The percent (%) increases in older women (≥75 years of age) than in younger women (≤29 of age) are: Ala, 7.7; Arg, 22; Asn, 11; Cit, 26; Glu, 44; Gln, 7.2; Lys, 10; Orn, 15; Phe, 8; Pro, 12; and Tyr, 22; whereas the percent (%) decreases in older women (≥75 years of age) than in younger women (≤29 of age) are: Ser, 15; and Thr, 2.4.

^b Comparison of older adults of > 64 years of age with younger adults of < 50 years of age. This study involved 701 men and 490 women.

^c Regression analysis of 6055 persons (433 men and 5622 women) aged 17 to 85 years.

^d Regression analysis of 2162 adults (men and women) aged 32 to 81 years.

^e Comparison of older adults (men and women) of > 60 years of age with young adults (20–39 years of age).

^f Regression analysis of 590 older adults (273 men and 317 women) with an initial mean age of 63 years during a 7-year period.

^g Comparison of 44 older men who had a mean age of 62 years with 36 young men who had a mean age of 21 years.

^h Regression analysis of 1212 older adults (374 men and 838 women) with an initial mean age of 61 years during a 10-year period.

ⁱ Comparison of 11 older adults (7 men and 4 women) who had a mean age of 68 years with 8 young adults (4 men and 4 women) who had a mean age of 31 years.

^j Comparison among 32 older men (with a mean age of 71 years), 68 young men (with a mean age of 26 years), 42 older women (with a mean age of 72 years), and 72 young women (with a mean age of 25 years).

^k Comparison of 26 older adults (men and women) who had a mean age of 70 years with 26 young adults (men and women) who had a mean age of 26 years.

^l Comparison of 5 older adults (men and woman) who had a mean age of 72 years with 5 young adults (men and women) who had a mean age of 30 years.

^m Comparison of 17 older adults (men and woman) who had a mean age of 65 years with 10 young adults (men and women) who had a mean age of 23 years.

↑ and ↓ indicate statistically significant increase and decrease, respectively, in older than in younger persons; “–-” denotes no statistically significant change; ♀, women; ♂, men.

Cit, citrulline; Hyp, 4-hydroxyproline; NR, not reported; Orn, ornithine; Tau, taurine.

There were conflicting reports regarding changes in plasma AA concentrations between young and older humans possibly due to differences in ages, food intakes, or both among free-living study participants. For example, compared with young men (the mean age of 25 years) and women (the mean age of 26 years), respectively, among all the so-called nutritionally essential AAs (EAAs) analyzed, the post-absorptive (over-night fasting) plasma concentration of only tryptophan in older men (the mean age of 71 years) was decreased by 13%, and the post-absorptive plasma concentrations of only branched-chain AAs in older women (the mean age of 72 years) were increased by 20–22% (Caballero, Gleason, and Wurtman 1991). There were no changes in other EAAs between young and older individuals (Achermann and Kheim 1964). Changes in plasma concentrations of NEAAs (including arginine, glutamine, and serine) in young vs older adults were not reported (Caballero, Gleason, and Wurtman 1991), possibly due to the previous ignorance of the nutritional and physiological roles of NEAAs in humans. By contrast, Achermann and Kheim (1964) found that the concentrations of the following AAs were lower in older men and women (the mean ages of 67 to 69 years) than those in young men and women (the mean ages of 30 to 32 years): alanine, −11%; arginine, −14%; branched-chain AAs, −13%; glycine, −11%; lysine, −17%; phenylalanine, −15%; serine, −18%; and tyrosine, −14%). These AAs are essential substrates for the syntheses of not only proteins but also other molecules [e.g., NO from arginine; glutathione from glycine; phosphatidylserine (a signaling molecule in the cell membrane) from serine; catecholamines and thyroid hormones from tyrosine; and creatine from arginine and glycine] with enormous physiological importance (Wu 2022). For example, these substances promote microvascular perfusion, anti-oxidative responses, DNA synthesis, fatty acid and glucose oxidation, energy provision, and physical contractions in skeletal muscle (Wu 2022). Chronic reductions in plasma AA concentrations [that can affect intracellular AA concentrations (Wu 2022)] and tissue sensitivity to insulin (Moore et al. 2015) may contribute to sarcopenia in adults. Some investigators continue to ignore the dietary intake (Church et al. 2020; Park et al. 2021; Pinckaers et al. 2023) or plasma concentrations (e.g., Church et al. 2020; Park et al. 2021; Pinckaers et al. 2022a) of NEAAs in aged humans.

Concentrations of free AAs in the skeletal muscle of young vs older adult humans

At present, little information is available regarding concentrations of AAs in the skeletal muscle of elderly vs young adult humans, possibly because of the previous notion that muscle protein synthesis is regulated by extracellular, but not intracellular, AAs in healthy young adults (Bohé et al. 2003). However, neither dietary intakes nor plasma/serum concentration of NEAAs in humans were determined in the study of Bohé et al. (2003). Results of studies over the past 20 years have shown that some traditionally classified NEAAs (e.g., arginine, glutamine, and glycine) can activate the mTOR cell signaling to stimulate protein synthesis and inhibit protein breakdown in skeletal muscle cells (Wu 2022). Of particular note, free AAs in the intracellular pool [with their concentrations being affected by their transmembrane uptake and release, intracellular synthesis and catabolism, and intracellular proteolysis (Wu 2022)], rather than free AAs in the plasma or serum, are the immediate precursors for protein synthesis. In addition, free NEAAs (Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, Pro, Ser, and Tyr) are much more abundant than free EAAs (His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) in the skeletal muscle of healthy adult humans, with the ratio (mol/mol) of free NEAAs to free EAAs being approximately 10:1 (Bergstrӧm et al. 1974). Thus, it is imperative that the intramuscular concentrations of all AAs (including both NEAAs and EAAs) be determined to provide insights into the availability of AAs for muscle protein synthesis.

We are aware of only one report of concentrations of most proteinogenic AAs in the skeletal muscle of elderly vs young adults (Gheller et al. 2021). These authors found that among all the measured proteinogenic AAs (albeit an incomplete list), only serine exhibited a reduced concentration in 60–80-year-old humans (n = 15) than 20–45-year-old adults (n = 43; Gheller et al. 2021). By contrast, a limited number of studies revealed lower intramuscular concentrations of some AAs (including β-alanine, glycine, histidine, 4-hydroxyproline, serine, threonine, and tryptophan) in old than in young rodents (Supplemental Table 1). Because intracellular AAs participate in multiple metabolic pathways including protein synthesis and its regulation, more studies should be conducted to determine intramuscular AAs in elderly vs young adults.

Concentrations of free AAs in the plasma (or serum) of sarcopenic vs nonsarcopenic adult humans

There are large variations in reports regarding changes in concentrations of plasma (or serum) AAs in sarcopenic vs non-sarcopenic adult humans (Table 2), likely due to differences in age, food intake, and health status. For example, compared with nonsarcopenic adults, concentrations of asparagine in the plasma (or serum) of sarcopenic adults were either increased (Calvani et al. 2018), decreased (Duan et al. 2023), or unchanged (Marques et al. 2023); concentrations of glutamine in the plasma (or serum) of sarcopenic adults were either decreased (Duan et al. 2023) or unchanged (Marques et al. 2023); and concentrations of taurine in the plasma (or serum) of sarcopenic adults were either increased (Calvani et al. 2018) or unchanged (Yeung et al. 2022). Nonetheless, most studies revealed that concentrations of branched-chain AAs, lysine, methionine, phenylalanine, threonine, tryptophan, and tyrosine were lower in the plasma (or serum) of sarcopenic than in non-sarcopenic elderly adults (Table 2). Interestingly, some of these changes occurred in a gender-specific manner, possibly due to differences in food patterns, hormones, and AA metabolism. For example, compared with nonsarcopenic adults, concentrations of leucine and valine were reduced in the plasma (or serum) of sarcopenic men and women; concentrations of threonine and tryptophan were reduced only in the plasma (or serum) of sarcopenic men, whereas concentrations of glutamate, histidine, isoleucine, methionine, phenylalanine, and proline were reduced only in the plasma (or serum) of sarcopenic women (Lo et al. 2021). Of note, most of these AAs are present in much lower amounts in plant- than animal-sourced foods (Li, He, and Wu 2021).

Table 2. Changes in the concentrations of amino acids in the serum or plasma of sarcopenic versus non-sarcopenic adult humans.

Amino acid	Serum		Plasma
	Calvani et al. (2018)^a	Yeung et al. (2022)^b	Duan et al. (2023)^c	Lu et al. (2020)^d	Marques et al. (2023)^e	Ottestad et al. (2018)^f	Toyoshima et al. (2017)^g	Lo et al. (2021)^h
Ala	–	NR	–	NR	–	–	–	– (♂); ↓ (♀. −14%)
Arg	–	–	↓ (–18%)	NR	–	NR	–	NR
Asn	↑ (+17%)	NR	↓ (–24%)	NR	–	NR	–	NR
Asp	↑ (+45%)	NR	–	NR	–	NR	NR	↓ (♂, −33%; ♀, −31%)
Cit	↑ (+22%)	NR	↑ (+23%)	NR	↑	NR	NR	NR
Cys	–	–	–	NR	–	NR	NR	NR
Glu	↑ (+32%)	–	↑ (+28%)	NR	–	NR	–	– (♂); ↓ (♀, −37%)
Gln	NR	–	↓ (–17%)	NR	–	–	–	NR
Gly	–	NR	NR	NR	–	NR	–	NR
His	–	NR	–	–	–	–	–	– (♂); ↓ (♀; −7%)
Ile	–	↓ (♂; −4%)	–	↓ (–8%)	–	↓ (–11%)	–	– (♂); ↓ (♀; −9%)
Leu	–	↓ (♂; −4%)	–	↓ (–7%)	–	↓ (–14%)	–	↓ (♂, −11%; ♀, −11%)
Lys	–	NR	↓ (–11%)	↓ (–4%)	–	NR	–	–
Met	↓	–	–	↓ (–7%)	–	NR	–	– (♂); ↓ (♀; −11%)
Orn	–	↓ (♀; −6%)	–	NR	–	NR	NR	NR
Phe	–	–	↓ (–11%)	↓ (–4%)	–	–	–	– (♂); ↓ (♀; −8%)
Pro	–	–	↑ (+57%)	NR	–	NR	↑ (+26%)	– (♂); ↓ (♀; −11%)
Ser	–	–	↓ (–22%)	NR	–	NR	–	NR
Tau	↑ (+16%)	–	NR	NR	NR	NR	NR	NR
Thr	–	NR	↓ (–16%)	↓ (–7%)	–	NR	–	↓ (♂, −14%); – (♀)
Trp	–	↓ (♂; −3%)	–	–	–	NR	–	↓ (♂, −9%); – (♀)
Tyr	–	↓ (♂; −5%)	–	–	–	–	–	NR
Val	–	↓ (♂; −4%)	–	↓ (–8%)	–	↓ (–6%)	–	↓ (♂, −12%; ♀, −13%)

In all these studies except Ottestad et al. (2018), blood samples were obtained from study participants after an overnight fast.

^a Comparison of 38 sarcopenic persons (13 men and 25 women) with a mean age of 76 years versus 30 non-sarcopenic persons (14 men and 16 women) with a mean age of 75 years.

^b Comparison among 201 sarcopenic men (a mean age of 73 years), 984 non-sarcopenic men (a mean age of 71 years), 125 sarcopenic women (a mean age of 73 years), and 1300 nonsarcopenic women (a mean age of 72 years).

^c Comparison of 30 possibly sarcopenic persons (a mean age of 86 years) vs 36 non-sarcopenic persons (a mean age of 75 years).

^d Comparison of 87 sarcopenic persons (32 men and 55 women) with a mean age of 74 years versus 102 non-sarcopenic persons (38 men and 64 women) with a mean age of 73 years. Sarcopenia in adults was identified if all the three following criteria were met: a lower-limb strength of < 18 kg for men and < 16 kg for women; a gait speed of < 0.8 m/sec for both men and women; a muscle mass of < 7.0 kg/m² for men and < 5.4 kg/m² for women.

^e Comparison of 8 sarcopenic persons (6 men and 2 women) with a mean age of 81 years versus 14 non-sarcopenic persons (11 men and 3 women) with a mean age of 78 years.

^f Comparison of 90 sarcopenic persons (21 men and 69 women) with a mean age of 78 years versus 327 non-sarcopenic persons (178 men and 149 women) with a mean age of 74 years. Non-fasting venous blood samples were obtained from study participants (about 2 h and 21 min after consuming a meal).

^g Comparison of 28 sarcopenic persons (10 men and 18 women) with a mean age of 85 years versus 132 non-sarcopenic persons (46 men and 86 women) with a mean age of 76 years.

^h Comparison among 33 sarcopenic men (with a mean age of 87 years), 33 non-sarcopenic men (with a mean age of 82 years), 44 sarcopenic women (with a mean age of 82 years), and 43 nonsarcopenic women (with a mean age of 81 years).

↑ and ↓ indicate statistically significant increase and decrease, respectively, in older than in younger persons; “–-” denotes no statistically significant change; ♀, women; ♂, men.

Cit, citrulline; Hyp, 4-hydroxyproline; R, not reported; Orn, ornithine; Tau, taurine.

There are reports that the development of sarcopenia in adults is associated with dietary AA intake. For example, in adult humans, a low risk for sarcopenia is correlated with a high intake of BCAAs and tryptophan, whereas gait speed (an indicator of skeletal muscle function) is positively correlated with dietary intakes of all EAAs (Ma et al. 2022). By contrast, Ebrahimi-Mousavi et al. (2022) reported no association between dietary intakes of BCAAs and odds of sarcopenia in adults (Ebrahimi-Mousavi et al. 2022). This discrepancy may be explained by differences in the dietary intakes of total protein and other nutrients (e.g., lipids and carbohydrates). However, no attention has been paid to dietary intakes of NEAAs by sarcopenic adults or the roles of these AAs in either modulating muscle protein synthesis (Church et al. 2020) or mitigating muscle loss and dysfunction in adults (Ma et al. 2022; van Vliet et al. 2015).

Acute effects of single administration of AAs on muscle protein metabolism in adult humans

In nutrition research, effects of bolus AA ingestion on muscle protein metabolism in people are often determined in combination with resistance- or endurance-type exercise. The latter stimulates both protein synthesis and protein breakdown in skeletal muscle, with a net effect on protein balance depending on dietary AA intake (Phillips 2012; Yang et al. 2012a). Amino acids are the building blocks of proteins, and some AAs (e.g., arginine, glutamine, glycine, and leucine) are activators of the mTOR cell signaling pathway to promote the initiation of protein synthesis in skeletal muscle (Wu 2022). In addition, arginine enhances the flow of blood and therefore the uptake of nutrients into skeletal muscle. Thus, single administration of 4 g leucine, a mix of 6 g BCAAs or their α-ketoacids, 6.7–40 g EAAs, or a mixture of 13–40 g EAAs plus some EAAs to young or older adults increased the rates of synthesis of mixed and myofibrillar proteins by skeletal muscle at rest and following resistance training (Table 3). Specifically, compared with the isonitrogenous control (alanine), ingestion of 4 g leucine along with a complete liquid diet over a 5-h period increased the rate of myofibrillar protein synthesis in skeletal muscle by 56% in elderly men (Rieu et al. 2006). Likewise, single oral administration of 2 g leucine to young men activated the mTOR cell signaling and increased muscle myofibrillar protein synthesis (Holowaty et al. 2023). Of note, intravenous or oral administration of a group of EAAs (defined differently by various research groups) or a mixture of EAAs plus some NEAAs for 2–4 h increases muscle protein synthesis in both young and elderly adults (Biolo et al. 1997; Bohé et al. 2001; Cuthbertson et al. 2005; Mitchell et al. 2015b; Tipton et al. 1999b). However, little is known about the response of skeletal muscle to administration of individual or a group of NEAAs in young or elderly adults. In contrast to data on protein synthesis, available evidence shows little change in the rates of breakdown of intramuscular mixed or myofibrillar proteins with aging in humans when expressed per kg fat-free lean mass (Rennie et al. 2010). Furthermore, there are reports that the rates of degradation of mixed proteins in skeletal muscle do not appear to be affected by intravenous or oral administration of a group of EAAs (Biolo et al. 1997; Tipton et al. 1999b). Collectively, a reduction in intramuscular protein synthesis appears to be primarily responsible for the loss of skeletal muscle protein during aging and can be a major therapeutic target.

Table 3. Acute effects of single administration of amino acids (AAs) or metabolites on skeletal muscle protein metabolism and function in young and/or older adult humans^a.

Study subjects (n)	Age (mean or range)	Single administration of AAs or metabolites	Study conditions	Acute effects	References
Young adults (6)	29	0.15 g of a mixture of 16 AAs^b (3-h iv infusion) per kg BW	Rest and immediately after RE in response to AA	↑ PS under both conditions; No change in muscle proteolysis after receiving 3-h AA infusion; ↑ Leg blood flow by AAs and exercise	Biolo et al. (1997)
Young adults (6)	22	40 g of a mixture of 17 AAs^c or 10 EAAs (oral)	4.5 h after RE	↑ PS at a similar rate with both AA infusions; No change in muscle blood flow or muscle proteolysis	Tipton et al. (1999a)
Young adults (4)	24	13.4 g of a mixture of 8 EAAs (oral)^d	2 h after EAA ingestion in the 12-h fasted state	↑ Net protein balance across leg skeletal muscle	Tipton et al. (1999b)
Older adults (5)	72	AAs plus glucose	Basal and feeding	↑ Net protein balance across leg skeletal muscle; ↓ in older vs young people	Volpi et al. (2000)
Young adults (5)	30
Older adults (24)	70	10 to 40 g of a mixture of 9 EAAs^e	12-h fasted state or 3 h after the ingestion of EAAs	No change in basal rates of MyPS or PS between young and older adults; ↑ MyPS, PS and mTOR signaling after EAA ingestion (plateau = 10 g) in both age groups; lower responses in older vs young adults	Cuthbertson et al. (2005)
Young adults (20)	28
Older adults (2)	67	6.7 g EAAs (1.7 g Leu) or 6.7 g EAAs (2.7 g Leu)	Immediately before and after ingestion of EAAs	↑ PS with 2.7 g Leu to a similar extent in both elderly and young adults; ↑ Phe net balance across leg	Katsanos et al. (2006)
Young adults (2)	30
Older men (20)	70	52 mg Leu/kg BW (3.84 g)^f	5 h of continuous feeding	↑ MyPS (by 57%); no change in whole-body protein metabolism	Rieu et al. (2006)
Older women (7)	68	7.5 g EAAs	4 h	↑ PS	Dillon et al. (2009)
Young men (6) and women (8)	29	10 g EAAs (1.8 g Leu) or 10 g EAAs (3.5 g Leu)	3 h after ingestion	↑ P-mTOR, P-S6K1, and P-4EBP1; no change in Ub signaling or net protein anabolism in skeletal muscle	Glynn et al. (2010)
Young men (41)	30	8 g Cit-malate	RE training	↑ Athletic performance; ↓ Muscle soreness	Perez-Guisado and Jakeman (2010)
Older men (16)	70	15 g EAAs	Bolus or pulse (4 × 3.75 g)	↑ P-S6K1 with bolus but not with pulse; ↑ PS up to 2 h in bolus and beyond 3 h with pulse	Mitchell et al. (2015b)
Young adults (6–17)	18–35	1–6 g taurine	RE training	↑ Skeletal muscle strength	Waldron et al. (2018)
Older adults (15)	61
Older men (15)	70	3 g Leu + 1.5 g Ile+ 1.5 g Val	0-5 h	↑ MyPS within 2 h after intake; no effect between 2 and 5 h after intake	Fuchs et al. (2019)
Older men (15)	71	α-Ketoacids of Leu, Ile and Val^g	0-5 h	↑ MyoPS within 2 h after intake; no effect between 2 and 5 h after intake	Fuchs et al. (2019)
Older men (24)	67	15 g milk protein + 0 or 1.5 g Leu	After RE	Leu increases MyPS after co-ingestion with protein	Holwerda et al. (2019)
Young men (8)	23	2 g Leu	3 h after ingestion	↑ P-S6K1; ↑ MyPS	Holowaty et al. (2023)
Young adults (11)	21	5.6 g BCAAs	4 h after RE	↑ MyPS by 15% (within 4 h after BCAA intake and RE)	Jackman et al. (2023)

^a Healthy study subjects with various ages (years) were either young or older adults.

^b A mixture of AAs: Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val; as well as Ala, Gly, Pro, Ser, and Tyr. This mixture of AAs lacked Asp, Asn, Gln, and Glu.

^c Ten EAAs (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) plus 7 NEAAs (Ala, Asp, Gln, Gly, Pro, Ser, and Tyr). This 17-AA mixture lacked Asn, Glu, and Cys.

^d Eight EAAs (His, Ile, Leu, Lys, Met, Phe, Thr, and Val). This mixture of EAAs lacked Arg and Trp.

^e Nine EAAs (Arg, His, Ile, Leu, Met, Phe, Thr, Trp, and Val). This mixture of EAAs lacked lysine.

^f The leucine diet was also supplemented with 11.6 mg isoleucine and 6.8 mg valine per kg body weight to achieve a 100% increase in plasma leucine concentrations and maintain plasma isoleucine and valine concentrations at normal postprandial values. The control diet was supplemented with 71 mg alanine per kg body weight to provide the same amount of nitrogen as the leucine diet.

^g The α-ketoacids of leucine, isoleucine, and valine were 3, 1.5, and 1.5 g, respectively (2:1:1, g/g).

Abbreviations: 4EBP1, 4E-binding protein 1; mTOR, mechanistic target of rapamycin; P-mTOR, phosphorylated mechanistic target of rapamycin; P-S6K1, phosphorylated p70S6 kinase 1; PS, mixed protein synthesis in skeletal muscle; RE, resistance exercise; Ub, ubiquitin-proteasome system signaling; ↑, increase; ↓, decrease.

NEAAs are more abundant than EAAs in the proteins of human skeletal muscle, with their ratio being 55 to 45 (g/g; Wu 2023). Growing evidence indicates important roles for NEAAs (e.g., arginine, glutamine and glycine) in enhancing protein synthesis in mammalian skeletal muscle cells in vitro and protein accretion in the skeletal muscle of experimental animals (e.g., pigs; Wu 2022). However, based on a study with young adults who showed a net positive nitrogen balance across skeletal muscle after receiving 2-h oral administration of eight EAAs and no change in intramuscular concentrations of all measured NEAAs, Tipton et al. (1999b) concluded that NEAAs are not necessary to stimulate net muscle protein synthesis in healthy individuals. This conclusion, however, lacked direct evidence and should be reevaluated. The AA solution used by Tipton et al. (1999b) lacked Trp (an EAA that is not synthesized by mammalian cells) and all NEAAs but human skeletal muscle could still respond to the ingestion of the 8 EAAs with an increase in protein synthesis during a period of 2 h. Clearly, such a result should not be interpreted to indicate that the dietary intake of Trp or NEAAs is not essential for maximizing net muscle protein synthesis in a longer term. In the case of Trp, a decrease in its dietary intake by healthy humans from 0.2 to 0.1 g per day for 5 days resulted in a negative nitrogen balance (Rose, Lambert, and Coon 1954). Likewise, both young and elderly adults responded positively to 3-h oral administration of a mixture of EAAs lacking lysine with an increase in muscle protein synthesis (Cuthbertson et al. 2005). This result does not negate an essential role for dietary Lys in muscle or whole-body protein synthesis during a longer period. Indeed, a decrease in the dietary intake of lysine by healthy humans from 1.2 to 0.6 g per day for 4 days resulted in a negative nitrogen balance (Rose et al. 1955). Thus, an acute increase in muscle protein synthesis in an absence of NEAAs or even some EAAs from a mixture of administered AAs does not mean that these AAs are not nutritionally or physiologically essential for this metabolic event in humans during a long-term period (e.g., weeks, months, and years). In support of such a notion, dietary supplementation with intact protein [e.g., whey protein providing both EAAs and NEAAs (Park, Choi, and Hwang 2018)], but not EAAs alone (Kim et al. 2012), for 3 months increased skeletal muscle mass and strength in older adults without regular exercise training. These results indicate that a sufficient provision of dietary NEAAs (e.g., arginine, glutamine and glycine; Table 5) are crucial for the optimal responses of skeletal muscle metabolism and function to dietary EAAs in elderly individuals.

Table 5. Effects of short- and long-term supplementation with amino acids or their metabolites in young or older adult humans^a.

Study subjects (n)	Age (mean or range)	Dietary supple- mentation per day	Study periods and conditions	Effects	References
Older men (10)	59–72	0.3 g CrM/kg BW	7 days	↑ LMB; ↑ Skeletal muscle strength; ↑ Physical performance	Gotshalk et al. (2002)
Older adults (100)	> 65	12 g EAAs^b (as snacks)	3 months	↑ Skeletal muscle strength; ↑ walking capacity; ↑ myocardial function	Scognamiglio et al. (2005)
Older men (15) and 8 women (8)	75	10–20 g CrM	14 days	↑ Handgrip strength; ↑ Physical performance	Stout et al. (2007)
Older men (CrM, 13; CrM + Protein, 10)	59–77	0.1 g CrM plus 0 or 0.3 g protein/kg BW	10 weeks and RE (3 days/week)	↑ LMB; ↑ Skeletal muscle strength; ↑ Physical performance; ↓ Breakdown of myofibrillar protein; CrM + Protein > CrM in the measured variables	Candow et al. (2008)
Older men (5) and women (7)	67	2 × 11 g EAAs and Arg	Between meals; 16 weeks	↑ LMB; ↑ Physical performance	Børsheim et al. (2008)
Older women (30)	58–71	0.3 g CrM/kg BW	7 days	↑ LMB; ↑ Skeletal muscle strength; ↑ Physical performance	Gotshalk et al. (2008)
Sarcopenic elderly (41)	66–84	8 g EAAs at 10 am and 5 pm	18 months of supplementation	↑ LBM; ↑ Whole-body insulin sensitivity	Solerte et al. (2008)
Older men (21) and women (19)	76	2–3 g HMB + 5–7 g Arg + 1.5–2.25 g Lys + 0.1 g Vit C	1 year of supplementation	↑ Whole-body protein synthesis; ↑ Lean tissue mass	Baier et al. (2009)
	75
Older women (7)	68	2 × 7.5 g EAAs	3 months of supplementation	No change in total Akt, P-Akt, P-mTOR, P-S6K1, P-4EBP1, or muscle strength; ↑ PS; ↑ LBM	Dillon et al. (2009)
Older men (15)	71	3 × 2.5 g Leu (with meals)	3 months of supplementation	No changes in skeletal muscle mass or strength, insulin sensitivity, or plasma lipid concentrations	Verhoeven et al. (2009)
Older men (38) and women (39)	76	2–3 g HMB + 5–7.5 g Arg + 1.5–2.25 g Lys^c	12 months	↑ LBM	Baier et al. (2009)
Older men with type-2 diabetes (30)	71	3 × 2.5 g Leu (with meals)	6 months of supplementation	No changes in skeletal muscle mass or strength, insulin sensitivity, plasma [lipids], or muscle fiber type	Leenders et al. (2011)
Older men and women (20)	84	2 × 4 g EAAs (31% Leu)	8 weeks	↑ Nutrition status; ↑ Skeletal muscle function; ↑ quality of life	Rondanelli et al. (2011)
Older men (5) and women (3)	68	3 × 4 g Leu	2 weeks of supplementation	↑ P-mTOR, P-S6K1, and P-4EBP1; ↑ PS in the postabsorptive state	Casperson et al. (2012)
Older men (4) and women (8)	60–80	2 × 1.6 g β-alanine	12 weeks	↑ Carnosine in skeletal muscle; ↑ Skeletal muscle strength	del Favero et al. (2012)
Sarcopenic women (38–39)	79	2 × 3 g EAAs (no His; Leu-enriched)	3 months with or without RE	↑ Leg muscle mass and strength with RE; No effects without RE	Kim et al. (2012)
Older women (15)	63	0.08 g CrM/kg BW	12 weeks and exercise (3 days/week)	↑ LBM; ↑ Skeletal muscle mass and strength	Aguiar et al. (2013)
Older adults (11)	67	2 × 1.5 g Ca-HMB	Bed rest for 10 days, followed by 8 weeks of RE	Alleviating loss of LMB over a 10-day bed rest; no change in skeletal muscle strength after RE	Deutz et al. (2013)
Older men and women (19 to 21)	71	2 × 0.8 g or 2 × 1.2 g β-alanine	12 weeks	↑ Skeletal muscle function and strength	McCormack et al. (2013)
Older men (7) and women (5)	50–71	0.1 g creatine/kg BW	32 weeks and regular RE	↑ LBM; ↑ Skeletal muscle mass and strength	Candow et al. (2015)
Older men (11)	87	3 g EAAs + 800 IU Vit D + 6 g MCTG	3 months	↑ Grip strength; ↑ Walking speed; ↑ Skeletal muscle strength	Abe, Ezaki, and Suzuki (2016)
and women (27)
Older men (4) and women (4)	72	2 × 7.5 g EAAs (containing 20–40% Leu)	12 weeks	↑ LBM; ↑ Skeletal muscle strength	Ispoglou et al. (2016)
Older adults with hip fracture (49)	86	3.08 g HMB	∼ 42 weeks	↓ Loss of LMB; ↑ total protein in the plasma	Malafarina et al. (2017)
Young men (14)	20	2 × 3 g Leu	12 weeks and RE training	No change in LBM, skeletal muscle fiber type, or skeletal muscle strength	Mobley et al. (2017)
Older adults (7 men and 9 women)	72	3 g HMB + 14 g Arg + 14 g Gln	6 months without exercise training	↑ Skeletal muscle mass; ↑ LMB; ↑ Skeletal muscle strength	Ellis et al. (2018)
Older men and women (7)	61	2.4 g β-alanine	28 days	↑ Skeletal muscle function and strength	Furst et al. (2018)
Young adults (6) and older adults (8)	20	1.5–4 g taurine	2 weeks; and RE training	↑ Skeletal muscle strength	Waldron et al. (2018)
Older men (12)	59	0.1 g CrM/kg BW	8 weeks and high-velocity RE	↑ Leg muscle and lower-body strengths (CrM alone); Synergistic effects of CrM and RE	Bernat et al. (2019)
Older men (6) and women (18)	80–92	10 g Cit-malate	3 weeks	↑ LBM and skeletal muscle mass; ↓ White-fat mass in women	Bouillanne et al. (2019)
Older adults with sarcopenia or cancer cachexia (131)	75	14.8 g Arg + 14.8 g Gln + 2.6 g HMB	31 to 36 days	↑ Handgrip strength; ↑ Gait speed	Saka et al. (2019)
Sarcopenic men (6) and women (27)	66	2 × 3.6 g BCAAs	5 weeks	↑ Skeletal muscle mass; ↑ Gait speed and grip strength	Ko et al. (2020)
Older men and women (38)	72	0.3 g Gln/kg BW + 10 g maltodextrin	30 days	↑ Glutathione in tissues; ↑ Anti-inflammation; ↓ Oxidative stress	Almeida et al. (2020)
Older women (23)	69	10 g Gln + 10 g maltodextrin	30 days	↑ Glutathione in tissues; ↑ Skeletal muscle strength; ↓ Oxidative stress	Amirato et al. (2021)
Older men and women (22)	60–73	3 g Cit-malate	6 weeks	↑ Walking speed	Caballero- Garcia et al. (2021)
Older men and women (276)	> 65	1.3–3 g HMB	2–24 weeks; bedridden or sedentary	↑ Skeletal muscle mass, strength, and function; ↓ Skeletal muscle protein degradation	Costa et al. (2021)
Older men (3) and women (5)	74	1.33 mmol Gly and 0.81 mmol N-acetyl- cysteine per kg BW	24 weeks	↑ Glutathione in tissues; ↑ Skeletal muscle strength; ↑ Mit function; ↓ Oxidative stress and inflammation	Kumar et al. (2021)
Older obese women (13)	61	1.5 g taurine	16 weeks	↑ Antioxidant capacity	Abud et al. (2022)
Sarcopenic patients with COVID-19 (40 in each group)	> 18	3 g Arg + other nutrients^d	12 days for Arg group and 20 days for Control group	↑ Grip strength; ↑ Skeletal muscle mass; ↓ Period of hospital stay	Bologna and Pone (2022)
Oder men (20–42) and women (28–47)	48–84	5 g CrM/kg BW	10–52 weeks; RE (2–3 days/week)	↑ Skeletal muscle mass; ↑ Skeletal muscle strength	Candow et al. (2022)
	60–80
Older men (15) and women (8)	65	3 g Arg	6 weeks and RE	↓ Heart rate in women; ↑ Muscle function in women	Córdova-Martínez et al. (2022)
Obese older men (20) and women (25)	67	10 g Cit-malate	12 weeks and HITT	↑ Skeletal muscle strength; ↓ White-fat mass	Marcangeli et al. (2022)

^a Study subjects with various ages (years) were either healthy older adults or sarcopenic adults.

^b 12 g EAAs (3.8 g Leu, 2 g Lys, 1.9 g Ile, 1.9 g val, 1.1 g Thr, 0.4 g cystine, 0.4 g His, 0.3 g Phe, 0.2 g Met, 0.1 g Tyr, and 0.1 g Trp).

^c 2 g HMB, 5 g arginine and 1.5 g lysine per day for study subjects with a body weight of 68 kg; 3 g HMB, 7.5 g arginine and 2.25 g lysine per day for study subjects with a body weight of > 68 kg;.

^d Creatine, L-carnitine, aspartic acid, magnesium, selenium and vitamins C and E.

Abbreviations: BW, body weight; Cit, citrulline; CrM, creatine monophosphate; EAAs, a mixture of His, Ile, Leu, Lys, Met, Phe, Thr, and Val; 4EBP1, 4E-binding protein 1; HMB, β-hydroxy-β-methylbutyrate (a metabolite of leucine); HITT, high-intensity interval training; LBM, lean body mass; MCTG, medium-chain triglyceride; Mit, Mitochondrial; mTOR, mechanistic target of rapamycin; P-mTOR, phosphorylated mechanistic target of rapamycin; P-S6K1, phosphorylated p70S6 kinase 1; PS, mixed protein synthesis in skeletal muscle; RE, resistance exercise; ↑, increase; ↓, decrease.

Acute effects of single ingestion of protein on muscle protein metabolism in adult humans

Dietary proteins are classified as either “fast digestible” (e.g., whey protein), “slow digestible” (e.g., casein and proteins in well-cooked beef), or “intermediate digestible” (e.g., egg, soy, pea, and rice proteins) based on the speed of their digestion to release small peptides and AAs in the small intestine (Dangin et al. 2001, 2003; Bax et al. 2013). Compared with “slow digestible” proteins, the ingestion of “fast digestible” proteins usually results in more rapid appearance of AAs in the blood but a shorter period of elevated AA concentrations. Nonetheless, Park et al. (2021) reported that the bolus ingestion of 57 g beef, 57 g pork, or 2 eggs (equivalent to ∼11 g protein) increased whole-body protein synthesis and protein balance to a similar extent in healthy young adults. This result indicates that these animal proteins are equivalent for stimulating protein synthesis and inhibit protein breakdown in adults.

Much evidence has shown that, in young, middle-aged, and older adults, the rates of synthesis of mixed and myofibrillar proteins by skeletal muscle at rest and following resistance training are increased in response to a single ingestion of 10–40 g of animal (e.g., whey, casein, egg, or beef) proteins (Table 4). In young adults, a maximal increase in muscle protein synthesis occurred at the dietary intake of 20 g animal-sourced protein (Moore et al. 2009). There are reports that a 100% increase in the plasma concentrations of EAAs is associated with a 34% increase in the rate of muscle protein synthesis in young and older adults consuming animal-protein supplements (Church et al. 2020). Because of sustained increases in the circulating levels of all AAs, a single ingestion of 30 g milk protein (providing 6 g BCAAs) augmented muscle protein synthesis in older adults for a longer period of time than ingestion of 6 g BCAAs or their α-ketoacids (e.g., 5 vs 2 h; Fuchs et al. 2019). In addition, both young and older adults responded to a single ingestion of plant-sourced proteins, such as 20–40 g soy protein (Yang et al. 2012b; Mitchell et al. 2015a), 30 g potato protein (Pinckaers et al. 2022b), 60 g wheat protein, or 30 g plant protein blend (15 g wheat protein, 7.5 g corn protein, and 7.5 g pea protein; Pinckaers et al. 2022a), with an increase in plasma concentrations of EAAs as well as the synthesis of mixed and myofibrillar proteins by skeletal muscle. However, in contrast to 35 g casein, a single ingestion of 35 g wheat protein did not affect protein synthesis in the skeletal muscle of older adults (Gorissen et al. 2016), suggesting that the composition of dietary AAs plays a crucial role in this metabolic pathway. In addition, compared with 113 g soy, the bolus ingestion of 113 g beef (isonitrogenous to soy) had a greater effect on the synthesis of myofibrillar proteins in the skeletal muscle of middle-aged (∼55 years of age) men at both rest and after resistance exercise (Phillips 2012). Furthermore, Holwerda et al. (2019) reported that bolus co-ingestion of 15 g milk protein with 1.5 g leucine enhanced muscle protein synthesis following resistance exercise in older men, when compared with the consumption of the protein alone. Most recently, Pinckaers et al. (2023) found that the bolus ingestion of a beef-based meal as the primary source of protein (0.45 g protein/kg BW) stimulated muscle protein synthesis in older adults (65–85 years of age) to a greater extent than the consumption of an isocaloric and isonitrogenous vegan meal. Thus, an adequate amount of either animal or plant protein in diets can provide AAs to enhance muscle protein synthesis, but the effect appears to be greater with the intake of animal- than plant-sourced proteins.

Table 4. Acute effects of single ingestion of protein on skeletal muscle protein metabolism and function in young and/or older adult humans.

Study subjects (n)	Age (mean or range)	Single ingestion of protein	Study conditions	Acute effects	References
Young men (22)	25	30 g casein, WP, or free AAs	Over a period of 7 h	Leu retention in the whole body: Casein > WP > free AAs	Dangin et al. (2001)
Older men (8)	75	WP hydrolysate	30 min after RE	↑ PS by WP + Leu; no difference in	Koopman et al. (2006)
Young men (8)	20	plus leucine^a		PS between young and older men
Older adults (10)	70	30 beef protein	Postabsorptive state and 0–5 h after ingestion	↑ PS by 51% in young and older adults; plasma [AA] peaked at 100 min after ingestion in both age groups	Symons et al. (2007)
Young adults (10)	41	(113 g lean beef)
Young men (8)	22	18 g WP or SP	During and after RE	PS: WP > SP at 3 h after RE; no difference between WP and SP during RE	Wilkinson et al. (2007)
Young men (6)	22	0, 5, 10, 20 or 40 g egg protein	After RE	Peak plasma EAAs: 0.75–1 h for 5 g, 0.75–1.5 h for 10–20 g, and 0.75–2 h for 40 g; PS: ↑ from 0 to 20 g protein and reached a plateau at 20 g^b	Moore et al. (2009)
Older adults (17)	68	30 or 90 g beef protein	Postabsorptive state and 0–5 h after ingestion	↑ PS by 50% (with 30 g beef protein); no difference in PS between 30 and 90 g beef protein groups	Symons et al. (2009)
Young adults (17)	34
Young men (6)	23	∼22 g WP, SP, or casein (containing 10 g EAAs)	Rest and after RE	PS: WP > SP > casein at rest and after RE [within 3 h of ingestion)	Tang et al. (2009)
Older men (48)	74	20 g WP, casein, or casein hydrolysate	Rest	PS: WP > casein hydrolysate > casein	Pennings et al. (2011a)
Older men (24)	74	20 g casein	Rest and after RE	PS: no difference between young and older men at rest or after RE; ↑ in response to RE in both age groups	Pennings et al. (2011b)
Young men (24)	21
Men	55	113 g beef vs 113 g soy^c	Rest and after RE	↑ MyPS with RE; beef > soy for MyPS under both conditions	Phillips (2012)
Older men (30)	71	0, 20, or 40 g SP or WP	Rest and after RE	MyPS and plasma [Leu]: WP > SP under both conditions^d	Yang et al. (2012b)
Older men (14)	72	20 g WP or casein	Rest and after RE	↑ MyPS; WP > casein under both conditions	Burd et al. (2012)
Older men (15)	68	27 g WP	After RE	↑ P-mTOR and P-S6K1 in both age group; ↓ P-mTOR in older than young men after WP ingestion	Farnfield et al. (2012)
Young men (16)	22
Older men (33)	73	10, 20, or 35 g WP	Rest	↑ Muscle protein balance with dose	Pennings et al. (2012)
Older men (12)	75	20 g casein	0 to 6 h after protein ingestion	↓ Plasma [total AAs] at 1.5 and 2 h in older than young men; no difference in PS between the age groups at 2 and 6 h	Kiskini et al. (2013)
Young men (12)	21
Young men and women (9–10)	18–30	19 g SP + WP + casein; or 18 g WP	0 to 4 h after protein ingestion	↑ PS in both groups to a similar extent (0–2 h after ingestion); PS: SP + milk > WP (2–4 h after ingestion)	Reidy et al. (2013)
Older men (7)	59	0, 12, 24, and 36 g beef protein (0, 57, 113, and 170 g beef)	0–4 h after protein ingestion; both at rest and after RE	↑ MyPS with 36 beef protein at rest and after RE; No effect at lower doses	Robinson et al. (2013)
Men + Women (19 for WP; 22 for SP)	18–35	24 g whey protein vs 24 g SP	36 weeks and RE training	↑ LBM to a greater extent in the milk than the SP group; 3.3 vs 1.8 kg)	Volek et al. (2013)
Older men (46)	69	10, 20, 30, or 40 g WP	After RE	↑ Intramuscular [Leu] at 2 h: WP40 = WP30 > WP20 = WP10 > WP0; ↑ P-S6K1 in skeletal muscle	D'Souza et al. (2014)
Older men (9) and women (10)	69	20 g WP or DPP	After RE	↑ PS (for both WP and DPP); WP > DPP	Luiking et al. (2014)
Older men (33)	70	30 g WP or SP	After RE	↑ P-S6K1 in both WP and SP groups at 2 h after RE but only in the WP group at 4 h after RE	Mitchell et al. (2015a)
Older men (43)	71	0 to 40 g WP	Rest	MyPS: ↑ with protein intake; plateau at 0.25 and 0.6 g protein/kg LBM in young and elderly men, respectively	Moore et al. (2015)
Young men (65)	22	or egg protein
Older adults (40)	74	20 g casein	Rest	↓ PS in elderly vs young adults	Wall et al. (2015)
Young men (35)	22
Older men (15 in each group)	69	21 g WP + 3 g fat + 9 g Carb; 21 g WP; or 3 g fat + 9 g Carb	Post-prandial	↑ PS equally for 21 WP with or without fat + Carb, compared with fat + Carb	Kramer et al. (2015)
Older men (9–10)	55–75	30 g SP + WP + casein; or 30 g WP	After RE	↑ PS or net protein balance in both groups to a similar extent	Borack et al. (2016)
Older men (10)	69	30 g WP	Rest and after unilateral RE	↑ PKB and S6K1 activity by WP and RE; elderly < young adults	Francaux et al. (2016)
Young men (9)	22
Older men (12)	68–73	35 g wheat protein or casein, or 60 g wheat protein	0–4 h after ingestion	↑ PS for casein (0–4 h) and for 60 g wheat protein (2–4 h); no effect for 35 g wheat protein	Gorissen et al. (2016)
Sarcopenic men (15)	69	21 g WP	Post-prandial	↑ PS	Kramer et al. (2017)
Older women (11)	69	15 g milk protein with 4.2 vs 1.3 g Leu	4 h postmeal	↑ PS	Devries et al. (2018)
Older men (15)	72	30 g milk protein	0–5 h after ingestion	↑ MyPS (within 5 h after intake)	Fuchs et al. (2019)
Men (8)	22–24	56 g beef, 56 g pork, or 2 eggs	4 h postmeal	↑ whole-body PS; ↑ whole-body protein balance	Park et al. (2021)
Young adults (24)	24	30 g potato protein vs 30 g milk protein	0–5 h after ingestion; at rest and after RE	↑ PS for potato or milk proteins to a similar extent	Pinckaers et al. (2022b)
Young adults (12)	18–35	Plant protein blend vs milk protein^d	0–5 h after ingestion	↑ MyPS for plant or milk proteins to a similar extent	Pinckaers et al. (2022a)
Young adults (8 men/8 women)	72	Beef-based meal vs vegan diet^e	0–5 h after ingestion	↑ MyPS for both diets; beef-based diet > vegan diet	Pinckaers et al. (2023)

^a 0.16 g why protein hydrolysate plus 0.03 g leucine per kg body weight per h for 6 h.

^b Protein ingestion did not affect the phosphorylated levels of ribosomal protein S6 kinase 1, ribosomal protein S6, or the ɛ-subunit of eukaryotic initiation factor 2B in skeletal muscle.

^c Isonitrogenous amount of beef or soy.

^d Peak plasma Leu: 1.0–1.5 h for WP20, WP 40, and SP20; 1.5–2 h for SP40. Leu availability in plasma (indicated by the area under the concentration-time curve): WP40 = SP40 > WP20 = SP20 > WP0 = SP0. MyPS at rest: WP40 = WP20 > WP0, SP40 = SP20 = SP0, and WP > SP for 20 or 40 g protein. MyPS after RE: WP40 > WP20 > WP0, SP40 > SP0 = SP20, and WP > SP for 20 or 40 g protein.

^d Plant protein blend consisted of 15 g wheat protein, 7.5 g corn protein, and 7.5 g pea protein. Milk protein was 30-g milk protein concentrate. The content of glycine in milk protein was only 50% of that in the plant protein blend.

^e A beef-based meal consisted of 100 g lean ground beef, 200 g potatoes, and 150 g string beans as protein sources. A vegan diet consisted of 95 g soybeans, 95 g chickpeas, 95 g broad beans, and 200 g quinoa as protein sources. The two diets were isocaloric and isonitrogenous, providing the study participants with 0.45 g protein/kg body weight.

BW, body weight; Carb, carbohydrate; DPP, dairy product proteins; 4EBP1, 4E-binding protein 1; LBM, lean body mass; [Leu], leucine concentrations; mTOR, mechanistic target of rapamycin; MyPS, myofibrillar protein synthesis in skeletal muscle; P-mTOR, phosphorylated mechanistic target of rapamycin; P-S6K1, phosphorylated p70S6 kinase 1; PS, mixed protein synthesis in skeletal muscle; RE, resistance exercise; S6K, ribosomal protein S6 kinase 1; SP, soy protein isolate; WP, whey protein; ↑, increase; ↓, decrease.

A combination of animal and plant proteins in diets is effective in increasing muscle protein synthesis in humans. The rates of muscle protein synthesis did not differ in young adults within a 5-h period after the consumption of (a) 30 g milk protein or 30 g potato protein (Pinckaers et al. 2022b); and (b) 30 g milk protein or 30 g plant protein blend (15 g wheat protein plus 7.5 g corn protein plus 7.5 g pea protein; Pinckaers et al. 2022a). Likewise, rates of muscle protein synthesis in young adults (men and women) did not differ between the consumption of 19 g soy plus casein plus whey proteins and 18 g whey protein during the first 2 h after ingestion, but the effect of 19 g soy plus casein plus whey proteins was greater than that of 18 g whey protein during the last 2 h after ingestion (Reidy et al. 2013). Thus, a combination of whey, casein and soy protein can extend postprandial muscle protein synthesis for a longer period than whey protein alone, possibly due to a better balance of AAs (including arginine, methionine, and cysteine) in the mixture of animal and plant proteins. Interestingly, an adequate amount of animal and plant proteins has an equal efficacy in supporting protein synthesis for a few hours in the skeletal muscle of adult despite differences in the content of some AAs. For example, the content of the following EAAs was much lower in the plant protein blend than in milk protein (based on percentages of decrease): Ile, 33%; Lys, 65%; Met, 43%; Thr, 22%; and Val, 36% (Pinckaers et al. 2022a). The content of Trp is expected to differ substantially between the animal- and plant-sourced proteins (Li, He, and Wu 2021), but this AA was not analyzed by Pinckaers et al. (2022b, 2022a). These results indicate that some EAAs in diets may not be the major factors affecting protein metabolism within hours in the skeletal muscle of humans.

Effects of short- and long-term supplementation with AAs or their metabolites on skeletal muscle protein metabolism and health in adult humans

Results of extensive research has documented the beneficial effects of AAs and other nutrients in improving skeletal muscle health in the elderly (Table 5). They include total EAAs, individual or a mixture of BCAAs (particularly leucine), some individual NEAAs (e.g., arginine, glutamine and citrulline), a mixture of NEAAs (e.g., arginine plus glutamine; glycine plus N-acetyl-cysteine), AA metabolites [e.g., β-hydroxy-β-methylbutyrate (HMB, a metabolite of leucine), branched-chain α-ketoacids, taurine, β-alanine, anserine, carnosine, glutathione, creatine and carnitine], vitamins (e.g., vitamins C, D and E), and minerals (e.g., calcium, magnesium and selenium). For example, as reported for a single ingestion, oral daily administration of a mix of 7.2 g BCAAs (5 wk), 6.7–22 g EAAs (8–18 wk), 3 g EAAs with 800 IU vitamin D plus 6 g medium-chain triglyceride (3 months), or 10 g glutamine (for 30 days) increased lean muscle mass and muscle function (indicated by handgrip strength) in older adults, including sarcopenic individuals (Table 5). Similar results were reported for dietary supplementation with 3 g HMB plus 14 g arginine plus 14 g glutamine; for 6 months); 0.1 g glycine plus 0.132 g N-acetylcysteine/kg BW/day (for 24 wk), 3 g arginine plus antioxidants per day (for 6 wk), 3–10 g citrulline malate/day (an immediate precursor of arginine; for 3–6 wk), 1.6–3.2 g β-alanine/day (a precursor of carnosine; for 4–12 wk), and AA metabolites (Table 5). The latter include creatine monohydrate (0.1 g/kg BW/day for 32 wk, 0.3 g/kg BW/day for 7 days, or 0.08 g/kg BW/day for 12 wk), 10–20 g/day creatine monohydrate (for 2 wk), 3 g/day calcium-HMB (for 10 days to 42 wk), 1.3–3 g/day HMB/day (for 24 wk), or 2–3 g HMB plus 5–7.5 g arginine plus 1.5–2.25 g lysine per day (for 12 months). There is evidence that the concentration of HMB in the plasma of elderly people is much lower than that for young adults due to reduced leucine catabolism (Duetz et al. 2018). Notably, oral administration of 1.5 g/day taurine to adult humans for 16 wk enhanced the antioxidant capacity and health of skeletal muscle (Abud et al. 2022). Similarly, acute or chronic ingestion of 1 to 6 g taurine/day improved skeletal muscle strength and overall endurance performance in adult humans (Waldron et al. 2018). Among the AA metabolites with beneficial effects on skeletal muscle, taurine and creatine are abundant in meat (e.g., beef) but are absent from plant-sourced foods, whereas β-alanine is abundant in meat but is negligible in plant-sourced foods (Wu 2020). This indicates a major difference in nutritional values between animal- and plant-sourced foods for humans.

As noted previously, the single ingestion of leucine acutely stimulated muscle protein synthesis in humans (including older adults; Rieu et al. 2006). Similar results have been reported for the oral administration of leucine daily (3 × 4 g/day) for 2 wk (Casperson et al. 2012). However, long-term dietary supplementation with leucine (3 × 2.5 g/day) to older men with or without type-2 diabetes for 3–6 months had no effect on skeletal muscle mass or strength, whole-body insulin sensitivity, or plasma lipid concentrations (Verhoeven et al. 2009; Leenders et al. 2011). This indicates that the acute effects of a supplemental AA may not predict its long-term impacts on metabolism or health because protein balance in skeletal muscle depends on not only the rate of its protein synthesis but also the rate of its protein degradation. Of particular note, the rates of intramuscular protein turnover are regulated by a plethora of factors, including endocrine status, dietary intakes of AAs and other nutrients, physical activity, and ambient temperatures (Wu 2022). Because no single experiment has examined the roles of all the proteinogenic AAs (individual or mixtures) and related metabolites in mitigating human sarcopenia, available evidence does not allow us to provide any weighting of their importance. Nonetheless, the published studies underscore the nutritional and physiological significance of consuming well-balanced diets [consisting of whole foods of both animal and plant origins] that provide adequate amounts of all the needed nutrients.

Effects of short- and long-term supplementation with protein on skeletal muscle protein metabolism and health in adult humans

A short-term (e.g., 6-day) dietary supplementation with protein (15–42 g/day) enhanced muscle protein synthesis in older adults (Devries et al. 2018) but was not effective in alleviating their muscle loss (Dirks et al. 2014). By contrast, long-term supplementation with whey or milk protein (10–50 g/day) or the combination of milk and soy proteins increased lean body mass, as well as skeletal muscle mass and strength, while reducing white-fat mass in older adults (Table 6). In addition, co-ingestion with milk proteins (13–22 g) with 1.2–4 g leucine plus 120–800 IU vitamin D₃ per day, 20 g mixed proteins plus 800 IU vitamin D3 plus 300 mg calcium per day, or 11 g casein plus 11 g whey protein plus 7 g BCAAs plus 206 mg ursolic acid plus 432 IU vitamin D3 per day, for 30 days to 9 months, increased skeletal muscle mass and strength in older adults (including sarcopenic patients) (Table 7). Similar results were reported for sarcopenic women consuming milk plus EAA (–His) supplement for 3 months (Kim et al. 2012). Although whey protein had a greater effect on promoting muscle protein synthesis than casein (Table 4), these two types of protein had the same efficacy in increasing skeletal muscle mass and strength in adult humans during a period of 8 weeks (Wilborn et al. 2013). A meta-analysis of randomized controlled trials involving 1063 adults (≥ 18 years of age) revealed that in comparison with a low-fat diet with a standard amount of protein (providing 12–18% of energy), the consumption of a low-fat diet with a high amount of protein (providing 25–35% of energy) for a mean duration of 12.1 wk (≥ 4 wk) promoted the mobilization of fat tissue and reduced the loss of fat-free mass including muscle (Wycherley et al. 2012).

Table 6. Effects of short- and long-term supplementation with protein in young or older adult humans^a.

Aubertin-Leheudre et al. (2009)

Study subjects (n)	Age (mean or range)	Dietary supple- mentation per day	Study periods and conditions	Effects	References
Older men (9 for the meat group; 10 for the meat-free group)	51–69	Omnivorous diets with or without meat	12 weeks and RE training	↑ LBM, skeletal muscle mass, and the number of skeletal muscle fiber in meat-consuming individuals	Campbell et al. (1999)
Men (18 for milk protein; 19 for SP)	18–30	17.5 g milk protein vs 17.5 g SP	12 weeks and RE training	↑ LBM to a greater extent in the milk than the SP group (3.9 vs 2.8 kg)	Hartman et al. (2007)
Older women (21 for omnivores; 19 for vegetarians)	43–48	Plant proteins vs plant plus animal proteins	A mean of 12 years on either type of diet	LMB was greater in the plant + animal protein group vs the plant protein group (22.6 vs 18.2 kg)	Aubertin-Leheudre et al. (2009)
Older men (15) vs young men (16)	68	27 g WP	12 weeks and RE training	↑ P-mTOR and P-S6K1 in both age groups; ↓ P-mTOR in older than young men after WP ingestion	Farnfield et al. (2012)
	22
Older men and older women (34)	78	15 g milk protein	24 weeks	↑ Physical performance	Tieland et al. (2012a)
Older men and women (31)	78	15 g milk protein	24 weeks and RE training	↑ Physical performance; ↑ skeletal- muscle mass; ↓ body fat mass	Tieland et al. (2012b)
Adults (1063)	≥ 18	High vs standard amount of protein^b	12.1 ± 9.3 weeks (≥ 4 weeks); mean ± SD	↑ loss of fat tissue; ↓ loss of fat- free mass (including muscle)	Wycherley et al. (2012)
Young women (8)	20–21	24 g casein or 24 g whey protein	8 weeks and regular exercise	↑ Physical performance; ↑ skeletal- muscle mass	Wilborn et al. (2013)
Older men (11)	69	2 × 20.7 g WP	5 days of supplementation	No effect on immobilization-induced skeletal muscle disuse atrophy	Dirks et al. (2014)
Older men and women (60)	61	0.165 g milk protein per kg BW	24 weeks	↑ 0.45 kg LBM in the protein group; ↓ 0.16 kg LBM in the control	Norton et al. (2016)
	(50–70)
Older men (31)	72	15 or 30 g casein or soluble milk protein	10 days	↑ Myosin synthesis with 30 g protein regardless source and with 15 g soluble milk protein	Walrand et al. (2016)
Older women (11)	69	15 g protein (4.2 or 1.3 g Leu)^c twice	6 days; at rest and after RE	↑ MyPS; Effects: Milk > Milk + Soy; Exercise > Rest	Devries et al. (2018)
	(65–75)
Older adults (303 − 557)	55 (initial age)	High vs low animal protein^d	A mean of 9 years; rest and exercise	↑ lean mass; ↓ functional decline at both rest and regular exercise	Bradlee et al. (2018)
Older adults (156 − 449)	55 (initial age)	High vs low plant protein^e	A mean of 9 years; rest and exercise	↑ lean mass; ↓ functional decline only in persons with regular exercise	Bradlee et al. (2018)
Frail older adults (40)	77	9.3 g WP	12 weeks; no RE	↑ Skeletal muscle mass; ↑ Gait speed (muscle strength)	Park, Choi, and Hwang (2018)
Older men (47) and women (11)	69	31 g milk protein twice	12 weeks and regular exercise	↑ LBM; ↓ White-fat mass	Ten Haaf et al. (2019)
	(67–72)
Older men and women (22)	85	2 × 17 g milk protein (morning and evening)	10 weeks, and strength training 3 times/week	↑ Leg lean mass, thickness, knee extensor strength, and physical performance	Aas et al. (2020)
Older adults with sarcopenia (28)	≥ 65	12.8 g protein (with 8.5 g WP) + 1.2 g Leu + 120 IU Vit D	12 weeks; meal protein = 1.2–1.5 g per kg BW per day	↑ Gait speed	Lin et al. (2021)
Older men and women (61)	71	10.1 g milk protein	6 months and daily exercise training	↑ Skeletal muscle mass; ↓ White-fat mass	Nakayama et al. (2021)
Older men (9)	67	2 × 25 g WP (breakfast and lunch)	12 weeks and RE twice weekly	↑ Gait speed (WP alone); ↑ Muscle strength and LBM (RE alone); No synergistic effect for WP and RE	Griffen et al. (2022)

^a Study subjects with various ages (years) were either healthy older adults or sarcopenic adults.

^b Energy-restricted diets. Standard and high amounts of dietary protein provided 12–18% and 25–35% of dietary energy, respectively.

^c 15 g milk proteins containing 4.3 g Leu or 15 g milk plus soy proteins containing 1.3 g Leu.

^d Animal proteins were from a mixture of beef, lamb, pork, poultry, fish, and dairy. High- and low-protein intakes were 1.33 and 0.83 g/kg BW/day, respectively.

^e Plant proteins were from a mixture of legumes, soy, nuts, and seeds. High- and low-protein intakes were 1.33 and 0.83 g/kg BW/day, respectively.

Abbreviations: BW, body weight; EAAs, a mixture of His, Ile, Leu, Lys, Met, Phe, Thr, and Val; 4EBP1, 4E-binding protein 1; HMB, β-hydroxy-β-methylbutyrate (a metabolite of leucine); LBM, lean body mass; mTOR, mechanistic target of rapamycin; P-mTOR, phosphorylated mechanistic target of rapamycin; P-S6K1, phosphorylated p70S6 kinase 1; PS, mixed protein synthesis in skeletal muscle; RE, resistance exercise; SD, standard deviation; SP, soy protein; WP, whey protein; ↑, increase; ↓, decrease.

Table 7. Effects of short- and long-term supplementation with protein + amino acids + other nutrients on older adult humans^a.

Study subjects (n)	Age (mean or range)	Dietary supplementation per day	Study periods and conditions	Effects	References
Sarcopenic men (64) and women (120)	77	21 g WP + 3 g Leu + 800 IU Vit D either twice or once	13 weeks	↑ Skeletal muscle mass	Bauer et al. (2015)
Older obese men (14) and women (16)	64	21 g WP + 3 g Leu+ 800 IU Vit D	13 weeks and regular RE	↑ Skeletal muscle mass; ↑ Handgrip strength; ↑ Walking speed	Verreijen et al. (2015)
Sarcopenic men (53) and women (77)	80	22 g WP + 4 g Leu+ 100 IU vitamin D	12 weeks and physical activity (exercise)	↑ 1.7 kg LBM; ↑ Handgrip strength	Rondanelli et al. (2016)
Older men (24)	71	21 g WP + 3 g Leu+ 800 IU Vit D	6 weeks (once/day before breakfast)	↑ PS; ↑ Skeletal muscle mass	Chanet et al. (2017)
Older women (11)	69	15 g milk protein with 4.2 vs 1.3 g Leu two times	6 days	↑ PS; ↑ Skeletal muscle mass; ↑ Skeletal muscle function	Devries et al. (2018)
Sarcopenic older adults (17–22)	86	21 g WP + 3 g Leu+ 800 IU Vit D twice	9 months and care home	↑ Skeletal muscle mass; ↑ Handgrip strength	Dimori et al. (2018)
Sarcopenic men (67) and women (112)	77	21 g WP + 3 g Leu+ 800 IU Vit D twice	13 weeks	↑ Skeletal muscle mass; ↑ Skeletal muscle strength	Verlaan et al. (2018)
Sarcopenic men (50) and women (25)	67	21 g WP + 3 g Leu+ 800 IU Vit D twice	30 days and care home	↑ Skeletal muscle mass; ↑ Handgrip strength; ↑ Walking speed	Barichella et al. (2019)
Sarcopenic men (62) and women (113)	78	21 g WP + 3 g Leu + 800 IU Vit D twice	13 weeks and care home	↓ Inflammation	Liberman et al. (2019)
	77
Sarcopenic men (17) and women (46)	82	21 g WP + 3 g Leu+ 800 IU Vit D twice	12 weeks and physical activity	↑ Skeletal muscle mass; ↑ Handgrip strength; ↑ Cognitive function	Rondanelli et al. (2020)
Older adults	50–80	20 g mixed proteins^b + 800 IU Vit D +300 mg Ca twice	12 weeks	↑ LBM	Kang et al. (2020)
Older men (18) and women (22)	75	11 g casein + 11 g WP + 7 g BCAAs + 206 mg ursolic acid + 432 IU Vit D	12 weeks	↑ LBM; ↑ Walking speed; ↑ Function of skeletal muscle mitochondria	Grootswagers et al. (2021)
Older adults with sarcopenia (28)	≥ 65	12.8 g protein (with 8.5 g WP) + 1.2 g Leu + 120 IU Vit D	12 weeks; Meal protein = 1.2–1.5 g per kg BW per day	↑ Gait speed	Lin et al. (2021)

^a Study subjects with various ages (years) were either healthy older adults or sarcopenic adults.

^b 50% casein + 40% whey protein + 10% soy protein. 20 g of this protein mix contained 3 g Leu.

Abbreviations: BW, body weight; EAAs, a mixture of His, Ile, Leu, Lys, Met, Phe, Thr, and Val; HMB, β-hydroxy-β-methylbutyrate (a metabolite of leucine); LBM, lean body mass; PS, mixed protein synthesis in skeletal muscle; RE, resistance exercise; WP, whey protein; ↑, increase; ↓, decrease.

Several lines of evidence indicate that long-term supplementation with milk protein has a greater effect than soy protein to sustain skeletal muscle mass in adult humans (Table 6). First, compared with the same amount of soy protein, daily dietary supplementation with 17.5 g milk protein (including casein and whey) to young men during a 12-week resistance exercise program increased lean body mass by 1.1 kg (Hartman et al. 2007). Second, dietary supplementation with 24 g whey protein per day to young men during a 36-week resistance exercise program enhanced lean tissue gain by 1.5 kg than those consuming 24 g soy protein (Volek et al. 2013). Third, long-term consumption of diets containing both animal- and plant-sourced proteins for a mean of 12 years resulted in a 4.4 kg greater lean body mass in middle-aged women, compared with the consumption of plant-sourced foods without animal protein (Aubertin-Leheudre and Adlercreutz 2009). Fourth, consumption of meat-containing omnivorous diets contributed to greater gains in skeletal muscle mass in older men during a 12-week resistance training program, compared with meat-free lactoovovegetarian diets (Campbell et al. 1999). Fifth, in most of the home-bound elderly, consuming < 65% of total protein from animal-sourced foods results in the deficiency of at least one EAA (based on the recommended requirement of 0.8 g protein/kg BW/day) and therefore protein undernutrition (Dasgupta, Sharkey, and Wu 2005). Although there are differences in nutritional quality between animal- and plant-sourced proteins, they should be provided in appropriate amounts and proportions to meet the dietary requirements of humans for all AAs and to prevent an excessive intake of some AAs (e.g., methionine and cysteine) from animal protein alone.

Older adults have higher requirements for dietary protein than currently recommended to maintain muscle mass and mitigate sarcopenia

As noted previously, AAs not only activate the mTOR cell signaling required for the initiation of mRNA translation but also serve as the building blocks of proteins and the precursors of antioxidative molecules in tissues including skeletal muscle (Dillon 2013; Wu 2022). Multiple AA-related factors likely contribute to sarcopenia in adults, including reductions in protein intake, plasma AA concentrations, tissue sensitivity to insulin, muscle protein synthesis, the endothelial synthesis of NO from arginine, and blood flow to skeletal muscle (Campbell et al. 2001, 2002; Cereda et al. 2022; Cruz-Jentoft et al. 2019; Lonnie et al. 2018; Murton 2015). In support of this view, aging is associated with the impairment of NO-dependent blood flow into skeletal muscle (Delp et al. 2008; Dillon 2013) and that either physical exercise or dietary arginine supplementation enhances the flow of blood (and thus AAs and other nutrients) to skeletal muscle in humans including 70-year-old men (Mitchell et al.0.2017; Wu et al. 2021). In addition, there is a relationship between sarcopenia and physical activity-associated blood flow, as exercise (aerobic or resistance training) can improve microvascular perfusion in skeletal muscle and can alleviate sarcopenia (Zhang et al. 2022). Furthermore, compelling evidence shows that effective mitigation of sarcopenia in adult humans requires protein intake that is at least 50% greater than the current recommended value (i.e., 0.8 g protein/kg BW/day; Paddon-Jones and Rasmussen 2009). Thus, elderly individuals consuming 1.2 g protein/kg BW/day lost 40% less lean body mass over a 3-year period than those ingesting 0.8 g protein/kg BW/day (Houston et al. 2008). Likewise, older persons (≥ 65 years of age) with minimal physical activity required 1.2 g of high-quality protein/kg BW/day to improve the strength and function of skeletal muscle (Morais, Chevalier, and Gougeon 2006). For elderly persons who are capable of exercise, sufficient intakes of AAs have a synergistic effect with appropriate physical activity to maintain or increase skeletal muscle mass and strength, which can be an effective countermeasure to sarcopenia (Aas et al. 2020). Adults with physical frailty who are not able to perform regular resistance training may also benefit from an increased intake of high-quality protein to alleviate muscle loss (Morley et al. 2010). At present, it is unknown whether all or certain AAs are responsible for the beneficial effects of dietary protein in mitigating the progression of muscle loss in adults. This knowledge is important for designing precise nutrition for adults because a chronic high intake of dietary protein would increase the whole-body production of ammonia and homocysteine (risk factors for renal disorders, brain damage and cardiovascular disease).

High risk for sarcopenia in vegans and vegetarians

Worldwide, less than 10% of the population are vegans (consuming no animal-sourced foods) and vegetarians (consuming dairy products and eggs but no other animal-sourced foods), and more than 90% of the population are omnivores. Based on a survey of people (aged 18 to 64 years in Canada and the United States but 16 to 64 years in other countries) in 2018, globally 3% and 5% of them were vegans and vegetarians, respectively, and 97% of them consumed animal products; in the United States, the same (3%) percentages of adults were vegans and vegetarians, and 94% of adults consumed animal products (Ipsos 2018). A large-scale investigation involving 30,251 generally health-conscious adults (79% women and 21% men) in the United Kingdom revealed that dietary protein intakes by vegans and vegetarians were 23% and 19% lower, respectively than those for meat eaters (Sobiecki et al. 2016 Supplemental Table 2). There is evidence that the incidence of sarcopenia in vegetarians is much higher than that in meat eaters (20% vs 6.2%, respectively; Wang et al. 2023). Similarly, vegan diets may increase risks for sarcopenia in older adults (Domić et al. 2022). Consequently, compared with meat eaters, vegetarians had higher risks for hip (+25%) and arm (+25%) fractures, whereas vegans had much higher risks for not only hip (+231%) and arm (+56%) fractures but also leg (+205%) and vertebral (+59%) fractures (Tong et al. 2020).

Roles of animal vs plant protein intakes in maintaining skeletal muscle mass and alleviating sarcopenia in older humans

Results of a recent systematic review and meta-analysis reveal a positive association between healthy dietary patterns and the maintenance of gait speed with age (an intermediate marker of sarcopenia risk) in adult humans (Van Elswyk et al. 2022). There is also interest in the role of dietary animal- and plant-sourced foods in maintaining skeletal muscle mass and mitigating human sarcopenia (Alexandrov et al. 2018; Bradlee et al. 2018; FAO 2007; Mariotti and Gardner 2019; Valenzuela et al. 2019; Nunes et al. 2022; van Vliet et al. 2015). Recent articles have highlighted experimental evidence indicating that animal-sourced foods are more effective than plant-sourced foods in improving muscle health and reducing risk for sarcopenia in adults (Bradlee et al. 2018; Domić et al. 2022; Tong et al. 2020). For example, in studying the effect of animal vs plant proteins with or without physical activity in older adults (both men and women) over a mean duration of 9 years, Bradlee et al. (2018) found that the intake of animal proteins (a mixture of beef, lamb, pork, poultry, fish, and dairy) resulted in a greater lean mass and a lower risk of functional decline (and thus sarcopenia), whereas the benefits of an equal amount of plant proteins (a mixture of legumes, soy, nuts, and seeds) were only visible in those who performed regular exercise.

Because eggs provide high quality protein which stimulates muscle protein synthesis, Smith and Gray (2016) have proposed that consumption of eggs can help mitigate sarcopenia in older humans. In support of this view, results of a randomized trial indicated that the consumption of a high-protein diet (1.4 g protein/kg BW/day) consisting of three whole eggs per day for 12 wk preserved skeletal-muscle mass in older adults (a mean of 71 years of age) with overweight or obesity, when compared with an isocaloric diet (1800 kcal/day) providing 0.8 g protein/kg BW/day (Wright et al. 2018). In addition, dietary supplementation with dried egg white (20 g protein/day) for 6 months improved upper body physical function in elder women (a mean of 74 years of age), in comparison with the isocaloric maltodextrin supplement (Ullevig et al. 2022). Most recently, Okamura et al. (2023) reported that consumption of ≥ 60 g egg/day resulted in a greater skeletal-muscle mass in elderly men with obesity and type-2 diabetes, as compared with control individuals ingesting < 60 g egg/day.

Consumption of fish has been suggested to improve muscle health and prevent in older people (Alexandrov et al. 2018; Leroy et al. 2023). This is likely but experimental data are lacking to date. Here, we use beef as a prototype of functional meat to reduce risk for sarcopenia in humans because of the following two reasons. First, among meats, beef is the only food with the known composition of all proteinogenic AAs and their functional metabolites (e.g., taurine, β-alanine, 4-hydroxyproline, anserine, carnosine, and glutathione; Wu et al. 2016). Dietary supplementation with these substances improves skeletal muscle strength and exercise performance in elderly adults (Waldron et al. 2019; Wu 2020). At present, there is no direct evidence for each of these AA derivatives in preventing or alleviating human sarcopenia. However, Eshima et al. (2023) reported that dietary carnosine supplementation to aging mice for 2 wk (1 week of pretreatment and 1 week during hindlimb unloading) enhanced skeletal muscle mass and strength.

Second, there are published studies regarding the effects of beef consumption in mitigating sarcopenia in older people, but we are not aware of such work involving pork or poultry meat (Table 8). Specifically, beef is an abundant source of all proteinogenic AAs in both adequate amounts and balanced ratios, while also providing physiologically essential nonproteinogenic AAs (taurine and β-alanine; Wu et al. 2016) as well as vitamins (e.g., vitamins B6 and B12) and minerals (e.g., iron, zinc, and selenium; Daly et al. 2015; Leroy 2023; Klurfeld 2024; Wu et al. 2014). The content and bioavailability of proteinogenic AAs in beef are greater than those in plant-sourced foods. In most of the studies involving effects of meat (e.g., beef) consumption on human health, their authors did not take, into consideration, bioactive and protective compounds (e.g., taurine, 4-hydroxyproline, creatine, and glutathione) present in whole foods (Leroy et al. 2022; Sekhar et al. 2011; Turner and Lloyd 2017). Taurine (a potent antioxidant) is essential for the integrity and functions of tissues, including skeletal muscle, heart and eyes, whereas β-alanine is required for the production of antioxidative and neuromodulatory dipeptides (carnosine and anserine). Furthermore, beef contains a large amount of creatine (essential for energy metabolism in tissues, particularly skeletal muscle and brain), as well as 4-hydroxyproline (a major precursor of glycine; Wu et al. 2016). There are myths that plants provide all nutrients that are available in animal-sourced foods. However, taurine, vitamin B₁₂, creatine, carnosine, and anserine are absent from plants, whereas β-alanine and 4-hydroxyproline are low or negligible in plants (Hou et al. 2019; Wu 2022). Thus, the bolus ingestion of ground beef (0, 57, 113, or 170 g) by middle-aged (59-year-old) men rapidly increased myofibrillar protein synthesis in their skeletal muscle in a dose-dependent manner, and the effect of beef was further enhanced with resistance exercise (Robinson et al. 2013).

Table 8. Effects of long-term supplementation with beef on maintaining skeletal muscle mass and alleviating sarcopenia in young and older humans.

Study subjects (n)	Age (mean or range)	Dietary supplementation per day	Study periods and conditions	Effects	References
Older men (47) and women (95)	73 (60–88)	1.1 to 109 g raw beef	12 months	↑ Skeletal muscle mass; ↑ nutritional status; ↓ blood cholesterol	Asp et al. (2012)
Older women (53)	60–90	220 g raw beef (45 g protein)	4 months and regular RE	↑ Lean body mass; ↑ skeletal muscle strength; ↓ circulating interleukin-6	Daly et al. (2014)
Older adults (men + women; obese; 26 in Control, 41 in beef group)	68	Beef supplement (30 g/3 times/day); vs regular diet^a	6 months	SPPB score: beef group > control group	Porter Starr et al. (2016)
Young men (10)	26	20 g beef protein after workout	8 weeks and regular RE	↑ Lean body mass; ↑ skeletal muscle mass; ↑ muscle strength performance	Naclerio et al. (2017)
Young men (5) and women (5)	19	46 g beef protein after workout	8 weeks and regular RE	↑ Lean body mass; ↑ skeletal muscle mass; ↓ body fat	Sharp et al. (2018)
Older men (29) and women (48)	71	220 g raw beef (45 g protein)	24 weeks and regular RE	↑ Lean body mass; ↑ skeletal muscle mass; ↑ muscle strength performance; ↑ global cognition function	Formica et al. (2020)

^a Protein intakes in the beef and control groups were 1.1 and 0.8 g/kg body weight/day, respectively. Both diets were 500 kcal deficit.

BW, body weight; EAAs, a mixture of His, Ile, Leu, Lys, Met, Phe, Thr, and Val; HMB, β-hydroxy-β-methylbutyrate (a metabolite of leucine); LBM, lean body mass; PS, mixed protein synthesis in skeletal muscle; RE, resistance exercise; SPPB, short physical performance battery; WP, whey protein. ↑, increase; ↓, decrease.

Third, long-term supplementation with beef to adult humans enhances their nutritional status, lean body mass, lower-limb muscle strength, and the duration of moderate physical activity (Table 8). For example, daily dietary supplementation with 46 g beef protein to young adults for 8 wk along with regular resistance exercise increased lean body mass and skeletal muscle mass, while reducing whole-body fat mass (Sharp et al. 2018). Similarly, daily dietary supplementation with 45 g beef protein to older women for 4 months along with regular resistance exercise increased lean body mass and skeletal muscle mass, while reducing the circulating level of interleukin-6 (an indicator of inflammation) in older women (Daly et al. 2014). Likewise, in older men and women (60–88 years of age), there were positive correlations between beef intake (1.1 to 109 g raw beef/day) and skeletal muscle mass (Asp et al. 2012). Furthermore, a higher protein intake from beef (30 g lean beef three times/day) improved physical strength and function in frail and obese older adults (Porter Starr et al. 2016). Thus, increasing the consumption of beef may provide an effective nutritional means to mitigate sarcopenia in adults.

Some epidemiological studies raised concern that the consumption of red meat (including beef) might increase risks for obesity, type 2 diabetes mellitus, and cardiovascular disease in humans (Willett et al. 2019). Note that 130-g raw beef contains ∼7.7 g lipids (Wu et al. 2016), which provide only 3.5% of the daily energy (2000 kcal) needed by a 70-kg adult. Lipids are an important part of a healthy diet for humans, and the consumption of some fatty acids (e.g., oleic acid and conjugated fatty acids) in beef increases high-density lipoproteins (HDL; “good cholesterol”) and decrease low-density lipoproteins (LDL; “bad cholesterol”) in the blood of men and women (Smith et al. 2020). Recently, Lytle et al. (2023) reported that the weekly consumption of five 115-g ground beef patties (containing either 5.6% or 23.6% fat, on a fresh basis; prepared from the beef pectoralis muscle) for 5 wk did not negatively affect vascular function in healthy adult men with a mean age of 40 years. Furthermore, compared with the 5.6%-fat patty group, study participants in the 23.6%-fat patty group had 11% greater flow-mediated dilation in the forearm and 3.7% lower resting systolic blood pressure (Lytle et al. 2023), possibly due to the enhanced production of NO by endothelial cells (Wu et al. 2021). Thus, an appropriate amount of meat (e.g., beef) can contribute to an AA-balanced, healthy diet for humans.

Conclusions

With the US population growing older, sarcopenia is increasing in prevalence. The older population suffers from an insufficient intake of protein (especially animal-sourced protein) and insulin resistance. Insufficient protein intake leads to lower concentrations of several AAs that are important for skeletal muscle metabolism and health. In this review, we systematically and comprehensively summarized, for the first time to our knowledge, nutritional research related to (1) concentrations of free AAs in the plasma (or serum) and skeletal muscle of young vs older adult humans, (2) acute effects of single ingestion of AAs or protein on muscle protein metabolism in adult humans, (3) effects of short- and long-term supplementation with AAs (or their functional metabolites) on skeletal muscle protein metabolism and health in adult humans, (4) higher requirements for dietary protein in older people than currently recommended to maintain muscle mass and mitigate sarcopenia, and (5) roles of animal vs plant protein intakes in maintaining skeletal muscle mass and alleviating sarcopenia in older humans. Specifically, BCAAs were found to be lower in sarcopenic adults compared to non-sarcopenic adults. High dietary AA intake is correlated with lower sarcopenia risk. Administration of a mixture of BCAAs and EAAs following resistance training was found to increase rates of protein synthesis by skeletal muscle. Rates of protein synthesis are also increased by ingesting 10–40 grams of animal proteins. Much evidence shows that long-term dietary supplementation with animal and plant sourced proteins can enhance muscle protein synthesis in older adults, but both should be provided appropriately. In addition, most of the work cited herein is based on single dietary protein in acute or short-term studies but humans normally do not consume a single source of dietary protein (certainly not for vegetarians) and the results of such studies may not indicate long-term outcomes. Overall, older adults may benefit from an increase in the consumption of high-quality protein such as beef to combat their risk of sarcopenia. Furthermore, we highlighted discrepancies in the measured variables among the published studies and the need to determine intramuscular concentrations of AAs in young vs older adults. Finally, we proposed that adult humans require both EAAs and NEAAs in diets for maintaining muscle protein mass and function and, therefore, for mitigating the age-related sarcopenia. The integrated findings and new thoughts of this article are expected to guide further research for improving muscle health in older individuals.

Authors' contributions

GW designed and supervised this writing project. WH, EDC, and GW wrote the manuscript. HRC and GW contributed to manuscript revisions. All authors read and approved the final manuscript.

Acknowledgments

We thank Dr. Shalene H. McNeill and Dr. Clara Lau-Rosenow at National Cattlemen's Beef Association (Centennial, CO) for helpful discussion on the impacts of dietary protein intakes on human nutrition and health.

Disclosure statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Additional information

Funding

This writing project was supported by the National Cattlemen's Beef Association. The funder had no influence on the preparation of the paper.