Animal, Plant, Collagen and Blended Dietary Proteins: Effects on Musculoskeletal Outcomes

Dietary protein is critical for the maintenance of musculoskeletal health, where appropriate intake (i.e., source, dose, timing) can mitigate declines in muscle and bone mass and/or function. Animal-derived protein is a potent anabolic source due to rapid digestion and absorption kinetics stimulating robust increases in muscle protein synthesis and promoting bone accretion and maintenance. However, global concerns surrounding environmental sustainability has led to an increasing interest in plant- and collagen-derived protein as alternative or adjunct dietary sources. This is despite the lower anabolic profile of plant and collagen protein due to the inferior essential amino acid profile (e.g., lower leucine content) and subordinate digestibility (versus animal). This review evaluates the efficacy of animal-, plant- and collagen-derived proteins in isolation, and as protein blends, for augmenting muscle and bone metabolism and health in the context of ageing, exercise and energy restriction.

Given the utility of animal-derived protein intake in young healthy populations, a number of large cohort studies have assessed the relationship between animal-derived protein intake and muscle health across age. For example, in a study by Alexandrov et al. [49], muscle mass estimates from 24 h urinary creatinine and analysis of food intake by questionnaires illustrated that increased intake of both total protein and animal protein were associated with increased creatinine excretion (i.e., higher muscle mass) in both young and older males and females. Similarly, data from the Framingham Offspring Study found that higher protein intake from animal sources (e.g., red meat, poultry, fish) was associated with a higher percentage muscle mass over a 9-year period in adults over the age of 50 years [1]. These findings point towards positive effects of animal protein sources for the maintenance of muscle mass across the lifespan.
With this in mind, determining the efficacy of animal-derived protein feeding for potentiating muscle health in older adults has been a key aim of several investigations. Indeed, many studies have demonstrated that dairy [50] and meat [41,51] protein sources stimulate MPS in older adults. To demonstrate, one short-term study assessing the effects of a moderate (30 g) versus large (90 g) serving of 90% lean beef on MPS in younger (~35 years) and older (~68 years) adults found that MPS similarly increased in both age groups in response to the moderate serving of protein, with no further increase seen with the larger serving in either young or older adults [41]. This data is suggestive of a ceiling effect in healthy rested individuals in response to a single serving of animal protein, in line with the aforementioned "muscle full" hypothesis [48]. Interestingly, this data (and others [51,52]) does not support the notion of "anabolic resistance", which states that ageing muscle displays attenuated protein synthetic responses to protein feeding (and exercise) [53]. This is in disagreement with several studies that have shown anabolic resistance in response to feeding with EAA [46,54] and animal-derived protein [55]. To demonstrate, a retrospective cross-sectional study found that older adults exhibited a blunted protein synthetic response following 20 g casein protein consumption, compared to their younger counterparts [55]. This particular study pooled multiple well-controlled trials with similar study designs, thereby accruing a large volunteer pool (compared to other similar studies), and thus provides strong evidence to support the existence of anabolic resistance in ageing [9,55]. Although the mechanisms underlying anabolic resistance remain to be fully elucidated, a suggested contributor is the rate of protein digestion and AA absorption, which may impact the postprandial availability of AA for MPS [9,56], whereby compared to slowly digestible proteins, more rapidly digestible proteins result in a greater postprandial stimulation of MPS [57]. Whilst it has been shown on multiple occasions that older adults ingesting 20 g whey protein increased MPS to a greater extent than those ingesting 20 g casein protein (which has slower digestion and absorption properties compared to whey protein), leucine content was higher in whey protein, which is more likely the key anabolic driver [58,59]. Additionally, pulse feeding, which results in lower and more gradual aminoacidemia and leucinemia compared to bolus feeding, elicited equivalent net muscle anabolism in older adults (compared to bolus), suggesting that the speed of digestibility does not affect MPS [60]. The matrix and texture of the feed, which can be a consequence of food processing (e.g., mechanical processing such as mincing [57]), is another factor modulating the digestion and absorption and thus, potentially the protein synthetic response to animal-derived protein [57]. To highlight, Pennings et al. found that compared to beef steak, minced beef was more rapidly digested and absorbed, thereby stimulating a more rapid release of AA into circulation and thus enhancing postprandial net protein balance in older (~74 years) males, however, no differences in MPS were observed [61]. Further, irrespective of the coagulation mode, gelation of milk reduces AA absorption and the postprandial rise in circulating AAs [62,63]. Other considerations to obtain optimal digestion, absorption and synthetic kinetics in the context of ageing are chewing efficiency [64], cooking temperature [65] and cooking time [57]. As such, digestibility may modulate the anabolic response to protein feeding but this proposition remains contentious and requires further thorough investigation. It is, however, without doubt that the EAA profile of animal-derived proteins (e.g., higher leucine content) largely accounts for the robust anabolic responses to these proteins.
In more chronic experimental designs, one study assessed the effects of a 12-week diet with or without dairy-rich protein supplements in older adults [66]. Over this period, both groups saw negative changes in muscle strength, but a greater loss was observed in the control group, suggesting that protein intake might offset functional decline. In addition, the dairy protein group increased appendicular lean mass, indicating that a dairy-rich diet may be an effective strategy to counteract muscle loss in older adults. However, in older females habitually consuming more than the protein RDA, an additional daily protein drink containing 30 g whey protein (with reported 87% compliance) had no effect on muscle mass or function over a 2-year period [67], suggesting that the effectiveness of dietary protein may depend on the nutritional status and habitual protein intake of older individuals.
In regard to exercise × protein interactions, animal-derived protein sources can enhance the magnitude and duration of the increase in MPS in both young and older adults [47,68], therein delaying the "muscle full" set point [47]. In order to maximise the MPS response to acute resistance exercise (RE), research has focused on optimising protein feeding strategies, albeit mostly in younger adults. For example, Witard et al. [44] found that ingestion of 20 and 40 g whey protein isolate increased myofibrillar MPS above 20 g at rest and after unilateral RE in young health males, with no difference in MPS stimulation between 20 and 40 g. This data indicates that 20 g of whey protein is sufficient to stimulate maximal MPS post-exercise in the young with doses > 20 g leading to AA oxidation and ureagenesis, at least in the case of unilateral RE [44]. Indeed, it is not just animal-derived whey protein that can promote exercise × protein interactions. The slowly digested protein, casein, which elicits prolonged hyperaminoacidemia (likely due to slow gastric emptying) [69], has been shown to stimulate myofibrillar MPS and anabolic signalling 1-6 h post-RE [70].
Although 20 g whey protein appears to saturate MPS in young individuals, older adults appear to be responsive to greater protein doses in the context of exercise. For example, Yang et al. [71] found that in older males performing unilateral leg RE, whole-body leucine oxidation increased in a dose-dependent manner with increasing amounts of whey protein isolate (0, 10, 20 and 40 g), with rates of post-RE MPS enhanced with the highest two doses. Further, increasing amounts of protein (0, 57, 113 or 170 g) derived from ground beef elevated myofibrillar MPS both at rest and after acute RE to a greater extent in middle-aged males (~59 years) [72]. Importantly, in older adults, the source of animal protein can influence exercise × protein anabolic responses. For example, a study in healthy older individuals [73] demonstrated that a single bolus of high whey protein (20 g whey protein, 3 g total leucine) consumed immediately after RE resulted in a higher rate of MPS 4 h post-exercise than with an isocaloric milk protein control drink (6 g milk protein). Similarly, whey protein was found to stimulate MPS to a greater extent than casein protein when combined with RE in older (~72 years) males [58]. Thus, the amount and source of animal-derived protein should be considered when looking to optimise age-related anabolic responses to acute exercise.
Repeated post-exercise increases in MPS culminate over time (i.e., in response to resistance exercise training (RET)), leading to gains in muscle mass and strength, which may be potentiated with protein-feeding across age. Indeed, a study comparing young and older males found that whey protein (26.2 g AA per serving) ingestion during 12 weeks RET increased mechanistic target of rapamycin (mTOR), a "master regulator" of muscle growth, both before and after RET in younger males (compared to exercise combined with placebo) [74]. However, in older males, there was an increase in whey protein plus exercise-induced mTOR protein phosphorylation before RET, but this was diminished after, perhaps suggestive of an effect of ageing on exercise and animal-protein interactions [74]. When assessing muscle mass and functional outcomes in mobility-limited older adults completing 6 months of progressive high-intensity RET, consuming 40 g whey protein daily had no greater effect on lean mass or strength than the isocaloric (but not isoproteic) control [75]. In contrast, a recent study by Kang et al. [76] reported that following daily whey protein (32.4 g) supplementation in frail older adults undergoing 12 weeks of RET, grip strength, chair-to-stand time and gait speed improved to a greater extent in the whey protein supplementation group than in the RET only group. This data suggests that animal-derived protein can positively influence muscle function. Whilst there are conflicting reports (as outlined above), a meta-analysis of 22 studies (6 of which included older adults) concluded that animal protein feeding potentiates muscle mass and function gains during RET across age [5]. Collectively, these reports indicate that animal protein supplementation when combined with exercise training may promote muscle mass and function, however, in older adults, the outcomes may depend on the protein dose, the duration of supplementation and/or the characteristics of the volunteers.
Hospitalisation, illness and/or advancing age can lead to a reduced appetite and a subsequent reduction in nutrient intake, leading to a hypoenergetic state and muscle loss [77]. This situation also presents during purposeful weight loss in the form of a reduced calorie diet, hence the need for optimal nutritional interventions that aim to preserve muscle mass and function in the face of energy restriction. In a recent study by Hector et al. [4], males and females aged between 35 and 65 years consumed either whey protein (27 g) or soy protein (26 g) supplements during a 14-day weight loss diet. Postprandial MPS was reduced less with whey protein than with soy protein (or carbohydrate (CHO) supplementation) after the intervention, which was predicted to be of importance for the preservation of muscle mass during longer-term energy restriction. Additional support for the use of whey protein during weight loss interventions comes from a study performed in overweight or obese older females on a reduced calorie diet (1400 kcal/d) [78]. During a 6-month intervention, participants received twice-daily whey protein (25 g per serving) supplements or the same does of CHO in the form of maltodextrin. Although no differences were seen in changes to lean mass or muscle strength between the groups, greater weight loss was achieved in the protein group, possibly a consequence of increased satiety and ensuing declines in energy intake [78,79]. In addition, relative to thigh volume changes, the protein group gained~6% more muscle than the CHO group [78]. In a separate study of older obese individuals on an 8-week weight loss diet, the addition of a 7 g whey protein supplement consumed five times daily did not enhance weight loss, nor did it significantly preserve lean mass [80]. There was however a greater increase in acute postprandial MPS with the protein group [80]. Thus, evidence to date suggests that animal-derived protein feeding during energy restriction can contribute to maintaining muscle health.
To summarise, dietary animal protein does appear to offer benefits to skeletal muscle health in terms of protein turnover, muscle mass and muscle function across the life-course and during both exercise and energy restriction interventions (Table 1). Higher protein intake associated with higher % muscle mass over a 9-year period Higher intake of animal protein had higher % muscle mass In those less active, only animal protein consumption reduced risk of functional decline Symons et al., 2009 [41] Healthy young adults (M n = 8, F n = 9, 35 ± 3 years) and older (M n = 10, F n = 7, 68 ± 2 years) randomly assigned to moderate or large protein serving (mean ± SD)

Bone
Given the importance of protein for bone turnover and matrix remodelling, particularly during growth and ageing [12,13], it is unsurprising that dietary protein has a critical role in modulating bone health. When assessing the effects of animal protein sources on phenotypic (e.g., mass) and functional (e.g., strength) outcomes related to bone health, two recent studies have both reported positive findings. In a cross-sectional study by Durosier et al. [82], bone mineral density (BMD), bone strength and distal radius and tibia bone microstructures were assessed in 746 older females (~65 years). There was a positive association between animal and dairy protein intake with predicted bone failure load (calculated as: force for which 2% of the bone would be loaded beyond 0.7% strain [83]) and stiffness of the distal radius and tibia, which was largely attributed to observed changes in the trabecular bone microstructures. A separate cross-sectional study using dietary intake questionnaire data from >1000 older males from the Osteoporotic Fractures in Men Study, showed positive associations between animal protein intake and bone strength [25]. The findings of each of these studies indicate beneficial effects of animal protein sources on bone strength in older adults.
As previously mentioned, it has also been suggested that diets rich in animal proteins could have negative impacts on bone health [15]. One hypothesis surrounding this relates to the greater acid-forming properties of meat and dairy foods, where it is thought that bone loss could occur through release of salts from the bone to balance the generation of acid [84,85]. Despite this, many studies have found no adverse effects of meat-based protein sources on urinary calcium excretion or other markers of bone health. For example, data from the Framingham Offspring Study found that in 615 older adults, lower protein intake overall was associated with increased bone loss over a 4-year period, while higher intake of animal protein was not associated with decreased BMD [86]. Similarly, in a randomised crossover study of healthy postmenopausal females that directly compared the effects of a high (20% of energy as protein) versus low (12% of energy as protein) meat diet on calcium homeostasis and bone turnover, it was reported that eating a high-meat diet for 16 weeks had no effect on urinary calcium excretion, retention or on circulating markers of bone turnover [87]. A further randomised crossover study also in post-menopausal females studied the effects of a low (10% of energy from protein) versus high (20% of energy from protein) protein and potential renal acid load (PRAL) diet for 7 weeks [88]. The high meat/high PRAL diet led to an increase in both the fractional rate of calcium absorption and urinary calcium excretion, while there was no change in markers of bone resorption or formation. While more evidence is required, these findings indicate that a diet high in animal protein does not adversely affect bone health.
The role of animal-derived protein intake and exercise-induced adaptations on bone health is less studied than protein intake alone. One study by Ballard et al. [89] included young males and females undergoing 6 months of RET and aerobic exercise training, who received a twice daily protein-containing supplementation (84 g/d total protein) or CHO. The protein group had higher plasma insulin-like growth factor-1 levels at the end of exercise training, while serum bone alkaline phosphatase (ALP) also increased with training and tended to be higher in those who received protein. The protein group also had higher concentrations of the bone turnover marker N-terminal telopeptide (NTx). Conversely, during RET in healthy young females, 10 days of high protein (in the form of 2.4 g/kg/d purified whey protein) supplementation during the end of 12 weeks of RET had no effects on bone metabolism, possibly a reflection of the short exercise and supplement period [90]. In relation to advancing age, a study by Holm et al. [91] saw postmenopausal females complete 24 weeks of RET with or without a 10 g whey protein-containing supplement (albeit with calcium and vitamin D) after each training session. The nutrient group had greater increases in BMD as well as increased bone formation (with increased osteocalcin) [91]. Although these effects cannot necessarily be attributed to higher animal protein per se (due to the multi-nutrient supplement), these findings suggest that beneficial effects on bone metabolism can be gained in older adults with long-term training and animal-derived protein provision.
It is generally understood that diet-induced weight loss can have adverse effects on bone health through increased bone resorption [12]. However, the effects of animal protein during weight loss on (markers of) bone health remains poorly studied. One double-blind, randomised, placebo-controlled trial addressed this via whey protein supplementation (20 and ≥40 g) during a combined resistance and aerobic exercise training program in obese/overweight adults [92]. In this study, whey protein, regardless of dose, had no effect on BMD or bone mineral content (BMC) during the intervention. A further trial studied overweight males and females undergoing 12 weeks of energy restriction (6-6.3 MJ/d) with a high-protein (27% of energy from meat, poultry and dairy protein) or standard weight loss diet (16% protein energy) [93]. In this trial, there were no differences in markers of bone turnover or calcium excretion between the groups. A separate study addressed whether a high dairy protein diet containing high calcium (~2400 mg/d) would influence bone turnover during energy restriction in overweight adults [3]. In this study, energy restriction decreased urinary calcium excretion regardless of the calcium content. Following the weight loss intervention, there was an observed increase in bone resorption (determined as an increase in the bone resorption marker deoxypyridinoline), in both groups; however, the diet high in calcium minimised overall bone turnover. Bone health biomarkers were also assessed in a study of pre-menopausal overweight/obese females given differing amounts of dairy protein (dietary protein 30% or 15% of energy) during diet-and exercise-induced weight loss [94]. There was an increase in C-terminal telopeptide of collagen type-I (CTX; a marker for bone turnover), osteocalcin (a marker for bone formation) and urinary deoxypyridinoline in the low (<2% energy from protein) and adequate (dietary protein 15% of energy) protein groups, while no changes in resorption markers but an increase in the bone formation marker amino-terminal pro-peptide of collagen I (P1NP) were seen in the high (30% energy from protein) protein group [94]. These studies indicate that high-protein diets, particularly when higher in calcium, may protect against bone loss during periods of energy restriction and weight loss. Further research is required to directly study the individual effects of protein and calcium on bone health.
In summary, diets high in animal protein appear to be beneficial for bone throughout the lifespan and may offer benefits to bone metabolism in older adults with exercise training. There is also evidence to suggest that animal protein, especially with calcium sufficiency, may counteract some negative effects that weight loss has on bone mass ( Table 2). Randomised crossover design Postmenopausal F (n = 16, 56.9 ± 3.2 years, mean ± SD) randomised to two diets: low protein, low potential renal acid load (PRAL) and high protein, high PRAL diet. Randomised, double-blind, placebo-controlled design Postmenopausal F were randomised to a protein-containing nutrient supplement (n = 13, 55 ± 1 years) or placebo (n = 16, 55 ± 1 years) in conjunction with 24-week RET (mean ± SEM) Nutrient supplement containing: 10 g whey protein, 31 g CHO, 1 g fat, 250 mg calcium and 5 µg vitamin D. 730 kJ in total. Placebo supplement containing: 6 g CHO and 12 mg calcium. 102 kJ in total. Supplements were consumed after each training session BMD, bone markers, dietary analysis Nutrient group had greater increase in BMD at the femoral neck than controls Increased bone formation and osteocalcin following training in nutrient group Randomised, double-blind, placebo-controlled design Obese/overweight adults were randomised to 0 g protein (n = 68, 50 ± 7 years) 20 g protein (n = 72, 48 ± 8 years) or ≥40 g protein (n = 46, 49 ± 8 years) combined with 36-week RET and aerobic exercise training 3 d/week for 36 weeks (mean ± SD) Unrestricted diet in combination with whey protein supplementation (0, 20, 40 or 60 g/d) (

Skeletal Muscle
Given the widely reported benefits of animal-derived protein sources on muscle health across the life course, as outlined in Section 2.1 (e.g., References [41,[49][50][51]), yet also considering the sustainability of animal-versus plant-derived protein [6], observational studies have assessed the relationship between plant protein consumption and the preservation of muscle health across age. One cohort observational study found that plant-derived protein intake was not positively associated with leg lean mass in older adults, but animal-derived protein was [49]. Additionally, a large (n = 2066) longitudinal cohort study of older adults (70-79 years) also showed the importance of protein quality and composition for maintaining muscle mass. In this study, plant protein was not related to a reduced loss of lean mass and appendicular lean mass, however, animal protein was [95]. Similar observations were also seen in a cross-sectional study by Sahni et al. [96] in a wide-ranging age-group (29-86 years), where plant protein intake did not positively associate with leg lean mass, but high total and animal protein intake did. In this study, quadricep strength was greater in the highest plant protein intake quartile comparatively to the lowest quartile, suggesting that sufficient plant protein intake may help to reduce age-related loss of strength.
Despite a lack of convincing evidence from observational cohorts in regard to plant-derived protein (versus animal) and muscle health, a number of studies have gone on to directly compare musculoskeletal-related physiological responses between plant-and animal-derived protein.
For example, in healthy young males, ingestion of whey protein stimulated MPS to a greater extent than soy protein, despite matched EAA content [97]. This phenomenon of a diminished MPS response also translates into ageing as Yang et al. [71] demonstrated that, in rested older males, ingestion of either 20 or 40 g whey protein increased MPS, while ingestion of either dose of isolated soy protein elicited no such increases. Furthermore, heightened rates of leucine oxidation were observed in response to ingestion of both 20 and 40 g of isolated soy protein, which may indicate AA oxidation [98]. Similar observations were shown in middle-aged males, whereby postprandial rates of MPS were lower after soy protein ingestion compared to beef [35]. Considering these findings, it may be that consumption of a greater quantity of plant-derived protein may be required to overcome the reduced anabolic response of this protein source. Indeed, a recent investigation by Gorissen et al. [99] in older males demonstrated that although 35 g wheat protein did not stimulate MPS to the same degree as equal amounts of whey or casein protein, when the sources were matched for leucine content (4.4 g), consumption of 60 g wheat protein resulted in a greater MPS response than 35 g whey protein. Interestingly, plasma leucine concentrations increased to a greater extent following whey protein ingestion, with a more gradual appearance of plasma AA after wheat consumption. Sustaining postprandial AA increases may be beneficial in older populations through continued increases in MPS [60], however the practical challenge of getting older adults to consume greater amounts of plant-derived protein to achieve this must be considered given the reported lack of appetite [100] and rapid satiety [101] in older age.
Interestingly, some types of plant-derived proteins (e.g., potato and quinoa) contain adequate amounts of all EAA [9] and thus may offer sufficient anabolic alternatives to animal-derived proteins. Indeed, a recent study in young women found that 25 g of potato protein twice daily for 2 weeks (1.6 g/kg/d total protein) increased integrated MPS above baseline at rest, with no increase observed in those consuming a control diet (0.8 g/kg/d total protein) [102]. Whilst this greater anabolic response could be attributed simply to the greater amount of protein, it still demonstrates the ability of potato protein to stimulate MPS above a baseline diet already containing the RDA of protein, at least in younger individuals. Further, since it is well recognised that plant-derived protein sources can have inferior anabolic properties compared to animal-derived protein sources, the notion of blending different plant-derived sources together (in order to exploit the favourable AA profile of each) has been suggested in order to improve the anabolic quality of plant-derived protein sources. Protein blends, including plant-plant protein blends, are discussed in more depth in Section 5.
Considering protein × exercise interactions, it has been shown that combining whey protein ingestion with RE/T capitalises upon postprandial stimulation of MPS responses, therein promoting gains in muscle mass and strength [71,97]. However, the efficacy of plant-derived (as opposed to animal-derived) protein to potentiate exercise-induced anabolism is less well studied. In the context of acute exercise, whey protein ingestion in conjunction with unilateral RE elicited a greater MPS response than that of an EAA-matched (10 g) soy protein in young males [97]. However, both whey and soy protein increased MPS rates to a greater extent than casein protein in both rest and exercise conditions. This may be due to the slower nature of casein digestion and subsequent aminoacidemia, with whey protein increasing aminoacidemia to a more rapid and greater degree than intermediary soy protein [97]. Conversely, comparable MPS stimulation was observed post-exercise in those consuming potato protein (25 g twice daily) and control diet groups over a 2-week period, highlighting the potency of RE as an anabolic stimulus [102]. In the context of ageing, a randomised cross-over study by Wilkinson et al. [103] found that the ingestion of soy protein (18.2 g) with acute RE increased MPS responses to a lesser degree than that of isonitrogenous whey protein in young males. This was despite greater total plasma AA and similar leucine concentrations following consumption of soy protein.
In regard to chronic exercise × plant-protein interactions, a 6-week whole-body RET (3 d/week) intervention involving supplementation of whey or soy protein (1.2 g/kg, consumed as three equal doses per day) increased lean mass and strength in young adults to a greater degree than an isocaloric maltodextrin placebo [104]. Furthermore, no differences between the protein groups were observed, and fractional breakdown rate remained constant throughout, suggesting greater MPS, independent of protein source. Conversely, some have reported no effects of whey or soy protein on muscular adaptations to RET [105] and others have shown that compared to milk, soy protein induced inferior gains in muscle hypertrophy in young males [106]. Interestingly, other plant-derived protein sources have demonstrated similar benefits when combined with RET. For example, twice daily ingestion of pea or whey protein (26.6 g protein, 2.9 g leucine and 23.9 g protein, 3.9 g leucine, respectively) combined with progressive upper-body RET each improved bicep thickness after 42 and 84 days in young males [107]. Furthermore, sub-analysis of weaker (at study start) adults showed that consuming pea protein increased muscle thickness to a greater degree than whey protein or placebo. Similarly, consumption of rice protein isolate (48 g protein, 3.84 g leucine, 3 ×/week) during 8 weeks of whole-body RET improved lean mass gains and body composition to a comparable extent as isonitrogenous whey protein (48 g protein, 5.5 g leucine) in young males [23]. In a study of older males, the addition of a diet high in beef or soy protein (0.6 g protein/kg/d from beef or soy, respectively) to whole-body RET for 12 weeks, each increased strength and m. vastus lateralis cross-sectional area to a similar extent [81]. Interestingly, the comparable increases in skeletal muscle mass independent of protein source in a number of these studies [23,81] may be a result of consuming greater protein amounts (and subsequently leucine), thereby offsetting the often reduced EAA content with plant-derived proteins and supporting augmentation of RET-induced gains in lean mass. Thus, sustained consumption of greater quantities of plant-derived proteins in conjunction with RET may be sufficient to support increases in muscle mass (Table 3).  No difference in total kcals or percentage fat intake between groups Protein intake was significantly greater in the potato protein group compared to control MPS increased above baseline at rest in the potato protein, but not control, group MPS increased similarly above baseline with exercise in both groups In response to exercise, total protein kinase B (PKB/Akt) increased compared to baseline Main effect of time for total mechanistic target of rapamycin and ribosomal protein s6 Abbreviations: AA, amino acids; BMI, body mass index; CHO, carbohydrate; CSA, cross-sectional area; EAA, essential amino acid, FBFM, fat-and bone-free mass; F, females; FBR; fractional breakdown rate; FSR, fractional synthesis rate; LM, lean mass; M, males; MPS, muscle protein synthesis; RE, resistance exercise; RET, resistance exercise training; RM, repetition maximum; SD, standard deviation; SEM, standard error of the mean.

Bone
Despite plant-derived proteins varying in AA composition depending on the plant protein source (i.e., corn and wheat), similar sulphur content has been reported across these sources [110] suggesting possibly ubiquitous effects on bone health. Although limited studies directly comparing the effects of protein sources on bone health are available, a recent meta-analysis by the National Osteoporosis Foundation was undertaken evaluating the influence of differing protein source supplementation on the bone health of healthy adults [111]. This analysis of randomised controlled trials concluded that supplementation of either soy or animal protein for >1 year was beneficial on multiple outcomes of bone health (BMD in lumbar spine, total hip, femoral neck and total body), with neither more advantageous than the other. Moreover, a separate randomised controlled trial by Dawson-Hughes et al. [2] showed that during a 3-year supplementation period of calcium and vitamin D in older males and females, greater protein intake (irrespective of the source) was associated with increased BMD. It is important to note that such improved bone-related outcomes resulting from increased protein intake require sufficient dietary calcium intake, and the relationship between protein intake and BMD was not observed in the control group for this study [2]. Despite these findings, translation of the results from these supplementation studies into additional clinically relevant outcomes remains unclear. For instance, one recent cohort study showed that greater protein intake of animal protein was associated with reduced risk of hip fracture in older males, whereas plant-derived protein was not [24], which could be related to the higher calcium content in animal-derived versus plant-derived protein sources. Comparatively, in a separate 5-year cohort study of older males and females, greater protein intake was associated with reduced fracture risk, but this was not related to protein source [112].
Variable findings on the effect of protein sources on bone health may result from additional constitutive elements present, such as isoflavones, which are predominantly present in soy protein products. In support of this, epidemiological studies have associated a decreased risk of bone loss and hip fracture risk in older Asian populations with consuming proportionally more soy protein [113]. Structurally similar to oestrogens, isoflavones have been demonstrated to reduce bone turnover through a combination of stimulating bone formation and inhibiting bone reabsorption [114,115]. Furthermore, isoflavone inclusion rather than protein alone may be an important aspect for bone health, as in a 24-week supplementation period in perimenopausal women, only supplementation with isoflavone-rich soy was able to attenuate losses in BMD and BMC when compared to isoflavone-poor soy protein or whey protein control [116]. However, results from isoflavone supplementation studies have been inconsistent, with supplementation of isoflavone-enriched products (110 ng/d) alongside habitual diets for 1 year not shown to prevent postmenopausal bone loss [117], suggesting that increased protein consumption may also be needed. That said, soy isoflavanols' (70 mg/d) supplementation increased bone formation markers (i.e., bone-specific ALP and osteocalcin), whilst reabsorption markers remain unchanged (i.e., CTX and NTx) [118]. Although further investigation is required to elucidate potential benefits of isoflavones and corresponding protein supplementation, high habitual soy protein intake (containing isoflavones) may be beneficial for the maintenance of bone health and/or the attenuation of bone loss.
To summarise, plant-derived dietary protein has the potential to induce similar anabolic responses to animal-derived protein, particularly when matched for leucine, in the context of acute and chronic exercise. Additionally, plant protein alone (i.e., not in the context of exercise/energy restriction) demonstrates beneficial effects on certain aspects of bone health (e.g., BMD), although this may be in part due to the effects of plant protein containing isoflavanols (Table 4). Randomised cross-over intervention study design Low meat soy supplemented vs. high meat n = 13 7 weeks, healthy postmenopausal females (F) (59.9 ± 5 years, mean ± standard deviation (SD)) Low meat soy supplemented-55 g/d meat, 25 g soy protein High meat-170 g/g meat All meals provided

Skeletal Muscle
Collagen proteins are the most abundant proteins in the human body [121], accounting for 25-30% of total protein body mass [26], and are the major constituents of many tissues, including connective tissue, tendons, ligaments and bones [122]. Thus, dietary collagen is likely a key mediator of musculoskeletal remodelling throughout the lifespan. As such, collagen supplementation, in the form of collagen hydrolysates or gelatin, has recently gained popularity as an alternative or adjunct protein source to animal-and/or plant-derived sources for maintaining or even potentiating muscle and/or bone health (i.e., mass/function). This may seem counterintuitive since dietary collagen is rich in non-essential amino acids (NEAA's; e.g., proline, glycine), low in EAA's (e.g., methionine, leucine) and lacks tryptophan, rendering a DIAAS of 0 [27]. Expectedly, this has led to some questioning the anabolic potential of dietary collagen, at least compared to high-quality protein sources such as whey protein, which contain high levels of leucine and have a DIAAS of >1 [123]. Nevertheless, pre-clinical models have shown collagen-specific peptides to offset disease-induced muscle wasting [124], inhibit age-related muscle oxidative decline [125] and promote muscle hypertrophy via increased mTOR signalling [122], therein demonstrating the anabolic potential of supplemental collagen-derived proteins. This, coupled with the fact that dietary collagen has superb digestibility and becomes rapidly bioavailable following consumption in humans [28,126], suggests that there is potential for dietary collagen to mediate human skeletal muscle and bone remodelling. However, to date, the effects of collagen supplementation on muscle health across age, in the absence of allied exercise, has been sparsely studied.
In regard to ageing, older females consuming the RDA of protein with collagen constituting approximately half of the total protein provided, preserved lean body mass and maintained nitrogen balance [127]. In contrast, those consuming a similar quantity of whey protein experienced a loss in body weight with no change in body fat (potentially indicating a decline in lean body mass) and an increase in nitrogen excretion [127]. Despite collagen being regarded as a low-quality protein (according to PDCAAS and DIAAS scores), the NEAA's it does contain either have a low molecular weight or possess more than one nitrogen atom (e.g., hydroxyproline, hydroxylysine), meaning the nitrogen content of collagen on a per gram basis is high [8], and possibly greater than whey protein [127], which may explain the ability of collagen to help maintain nitrogen balance.
The ability of collagen supplementation to potentiate exercise-induced muscle adaptations is more widely studied than the effects of collagen supplementation alone yet remains contentious with mixed results depending on the outcome measure. In regards to body composition, Kirmse et al. [128] observed an increase in fat-free mass after 12 weeks of RET plus 15 g/d collagen peptide supplementation, which was not observed in the placebo group. However, similar changes in cross-sectional area and muscle thickness across the whole cohort (i.e., both groups) suggest that greater myofiber hypertrophy cannot explain these changes. Other data shows blunted RE-induced increases in anabolic signalling (p70S6K) (collagen vs. whey protein) [129] and muscle sub-fraction (myofibrillar and sarcoplasmic) MPS (collagen vs. α-lactalbumin) [130], with collagen protein, albeit in the context of short-term (3-days) aerobic exercise. The lack of tryptophan and low methionine and leucine content [27] in dietary collagen may explain the non-hypertrophic responses when used in an unblended fashion (i.e., when not blended with other dietary protein sources). Instead, it has been suggested that increased connective tissue/extracellular matrix (ECM) remodelling may contribute to the favourable changes in fat-free mass that are observed [128]. This supposition is supported by data showing that gelatin supplementation increased collagen content in engineered ligaments [126]. Similar mechanisms may also underlie muscle functional responses, specifically muscle strength, where studies have shown collagen peptide supplements to have no effect on maximal voluntary contraction [29,128], but did speed-up recovery of countermovement jump performance following strenuous exercise [29]. Since ECM degradation can occur following exercise [131], it is plausible that the purported collagen-induced ECM remodelling (e.g., increased collagen synthesis) occurred, therein improving fast/reactive movements that have a heavy tendon component (i.e., countermovement jump) [128]. Further, the ability of collagen supplementation to facilitate the recovery of additional exercise performance measures, such as maximal voluntary contraction, following an intense period of short-term RET was similar to that of whey protein [132].
In the context of exercise and ageing, Zdzieblik et al. found that RET for 12 weeks combined with 15 g/d collagen peptide supplementation led to substantial increases in fat-free mass (+4.2 kg) and decreases in fat mass (−5.5 kg) in sarcopenic males [26]. However, similar intervention studies using potent and established nutritional (e.g., animal-derived protein [5]) and pharmacological (e.g., testosterone [133]) stimulators of muscle growth did not observe such marked increases in fat-free mass [134], leading some to question the findings of Zdzieblik et al. [134]. More recently, Jendricke et al. [135] also reported greater increases in fat-free mass and a greater decrease in fat mass following dietary collagen supplementation, supporting positive body composition changes in response to this form of supplement. In the absence of known mechanisms, it has been suggested that a reduction in adipocyte size may contribute to changes in fat mass [121], in addition to the already mentioned hypothesis of ECM adaptations contributing to fat-free mass gains. That said, collagen peptide supplementation in older females did not increase rates of integrated collagen (or myofibrillar) MPS above baseline or in response to two bouts of RE [27], contradicting the suggestion of impacts on ECM remodelling (at least in older females). Zdzieblik et al. [26] also proposed that collagen-induced creatine synthesis may underlie changes in fat-free mass, yet daily provision of arginine and glycine from collagen is small, and thus this has been refuted as a potential mechanism [134]. Collagen supplementation has also been tested in the context of blood flow restriction, an exercise modality shown to induce favourable changes in muscle mass in the context of low-intensity RET (~20-30% of 1 repetition maximum) [136,137]. In older males, collagen hydrolysate supplementation for 8 weeks adjunct to low-load blood flow restriction RET tended to increase muscle cross-sectional area (+6.7%) more than placebo (+5.7%). Although this was only reported as a trend (i.e., non-significant), this is likely due to the low participant numbers (n = 11 in each collagen and placebo group), and thus further studies are required to substantiate these findings.
In the only study of its kind (to date) involving collagen supplementation, energy restriction (500 kcal/d reduction) and subsequent energy restriction plus activity reduction led to incremental declines in myofibrillar MPS, which increased during return to habitual activity when supplemented (throughout) with 30 g whey protein but not with isonitrogenous and isoenergetic collagen protein [77]. In the same study, both energy and energy plus physical activity restrictions (≤750 steps/d) led to reduced lean body mass and leg lean mass, neither of which were mitigated with either collagen or whey protein supplementation, despite protein consumption amounting to twice the RDA for protein [77].
To summarise, dietary collagen does not appear to stimulate MPS in the context of ageing and/or exercise. However, there is evidence to suggest that it can promote favourable body composition and muscle functional adaptations when combined with exercise, independent of age, possibly mediated by ECM remodelling. It is therefore plausible that collagen protein provided simultaneously with nutritional stimulators of myofiber hypertrophy (i.e., animal/plant protein sources), may maintain and/or potentiate muscle health via dual mechanisms targeting both ECM and myofiber remodelling ( Table 5). Table 5. Collagen-derived proteins: effects on muscle in relation to age, exercise, energy restriction and source.

Reference Study Design Protein Composition Measurements Key Outcomes
Oikawa et al., 2020 [27] Double-blind, parallel group, randomised controlled trial (RCT) within-subject design (unilateral leg-rest, contralateral leg-resistance exercise (RE)) 22 healthy older female (F) (n = 11/group, 69 ± 3 years, mean ± standard deviation (SD)) Randomised to collagen protein or whey protein 2 ×/d for 6 d and unilateral RE twice during 6 d period Randomised, double-blind, placebo-controlled design 77 premenopausal untrained F were randomised to full body RET 3 ×/week for 12 weeks and collagen peptide (n = 40, 38.3 ± 8.7 years) or placebo (n = 37, 41.6 ± 6.9 years) (mean ± SD) Supplements taken daily for 12 weeks Randomised, double-blind, placebo-controlled design 25 M (24.2 ± 2.6 years, mean ± SD) were randomised to full body RET 3 ×/week for 12 weeks and collagen peptide (n = 12) or placebo (n = 13) Supplements taken daily for 12 weeks Hydrolysed collagen peptide-15 g/d Placebo-15 g/d noncaloric silicon dioxide Body composition, strength, proteome Collagen peptide is bioactive, demonstrated by increased circulating levels of hydroxyproline 2 h following collagen peptide ingestion Body mass and FFM higher in collagen peptide group versus placebo 221 higher abundant proteins identified in collagen peptide group versus on 44 in placebo (proteomic analysis) Upregulated proteins in the collagen peptide group mostly associated with protein metabolism of contractile fibres Hays et al., 2009 [127] Double-blind, randomised, cross-over design 9 healthy F (71 ± 1 years, mean ± standard error of the mean (SEM)) completed 2 × 15 d trials (7 d wash-out period in between) Each trial consisted of consuming 0.8 g protein/kg body weight/d with either whey protein or collagen peptide intended to providẽ 0.4 g/kg body weight/d Body weight decreased after whey but not collagen protein intake Nitrogen excretion was higher during whey versus collagen protein intake No difference in macronutrient intake between collagen peptide and whey protein groups (protein intake was 0.82 ± 0.04 g/kg/d) Double-blind, randomised, cross-over design 4 d wash-out 11 endurance trained adults (M n = 5, F n = 6, 24 ± 4 years, mean ± SD) engaged in daily high-intensity interval training with hydrolysed collagen or α-lactalbumin supplementation for 3 d Hydrolysed collagen peptides-60 g/d α-lactalbumin-60 g/d (breakdown of AA composition within each supplement can be found in original article)

Bone
The anabolic effects of collagen supplementation on bone health have been recognised in pre-clinical models demonstrating enhanced bone metabolism [138], microarchitecture [139] and offsetting age-related bone density decline [125], although the effects in humans are less well understood. In the context of ageing, post-menopausal females supplemented with 5 g/d calcium-collagen chelate (albeit with 500 mg calcium and 200 IU vitamin D 3 ) for 12 months had attenuated whole-body BMD losses compared to control (calcium and vitamin D 3 ) [140]. Whilst these positive effects cannot necessarily be attributed purely to collagen supplementation due to insufficient dietary control, these findings were later echoed by a 12-month randomised, double-blind, placebo-controlled trial, in which postmenopausal females given 5 g/d collagen peptide demonstrated significant increases in BMD of the femoral neck and lumbar spine and an increase in P1NP, indicative of an increase in bone formation [141]. Expectedly, the control group (5 g/d maltodextrin) displayed numerical (non-significant) declines in BMD and increases in CTX, indicative of bone degradation over the intervention period [141]. Since there were no differences in macro-or micro-nutrient intake observed between the treatment and control groups pre-or post-intervention, this may indicate that the positive effects on BMD are attributable to collagen supplementation.
In the context of exercise, it is thought that the liberation of collagen-specific AA (e.g., glycine, proline) from dietary collagen may potentiate exercise-induced collagen synthesis, therein facilitating bone remodelling. This hypothesis is somewhat supported by data from Shaw et al. [126], who found that supplementation with 5 g of vitamin-C (48 mg)-enriched gelatin (denatured form of collagen [122]) prior to intermittent high-impact exercise in young males increased circulating P1NP, indicative of increased bone collagen synthesis. Outwardly, this makes sense as vitamin-C is a cofactor for the enzymes lysyl hydroxylase and prolyl hydroxylase, which are essential for collagen synthesis [142]. In the absence of vitamin-C, 20 g/d collagen supplementation before and after strenuous exercise in young males had no significant effects on P1NP or β-isomerised CTX [29], suggesting that collagen alone does not support bone collagen synthesis. The discrepant findings between these studies may be due to the vitamin-C content or based on other methodological differences such as the differing collagen source (i.e., collagen peptide vs. gelatin) and/or amount (i.e., 20 g/d vs. 15 g/d vs. 5 g/d) of collagen provided.
The effects of collagen supplementation in tandem with chronic exercise training on bone health are not yet well established. In older sarcopenic males, 6 weeks of RET with 15 g/d collagen peptide supplementation did not augment RET-induced gains in bone mass [26], although the authors do not speculate about the reason for this lack of effect. Since one year of collagen supplementation alone (i.e., in the absence of RET) did improve BMD in older adults [141], it is conceivable that the supplementation period was too short to elicit collagen-induced effects in the slow turnover tissue that is bone.
Overall, dietary collagen appears to offer benefits to bone health in terms of offsetting age-related bone loss, potentially mediated by increasing bone formation and decreasing bone degradation. However, the synergistic effects of collagen supplementation and exercise remain contentious (Table 6).

Animal-, Plant-and/or Collagen-Derived Protein Blends
Protein blends offer a potentially viable and sustainable option to compensate for EAA deficiencies in some protein sources, thereby overcoming the inferior anabolic profiles of plant-and collagen-, versus animal-derived protein. Most research to date has assessed the effects of animal and plant blends in the context of skeletal muscle health, where the combination may exploit the digestive properties of each protein source, maximising AA availability and potentially extending and augmenting the MPS response [9]. For example, a protein blend containing 25% whey protein, 25% soy protein and 50% casein protein provided after acute RE in young healthy males stimulated mixed MPS in a similar fashion to whey protein [30]. Considering both supplements provided similar amounts of EAA and leucine, the similar anabolic stimulus is unsurprising and thus provides evidence to support the use of plant-derived proteins to promote acute muscle anabolism alongside animal-derived sources. This similar anabolic response to animal and plant blends (versus whey protein) also holds true in older adults [32]. However, it should be noted that in both of these studies, only 25% of the protein blend was plant-derived, and since plant-derived protein contains lower leucine and less EAA, the consumption of protein blends with higher percentages of plant-derived sources may not be as effective for stimulating muscle anabolism, particularly in older adults who display anabolic resistance [7,55], although this remains to be determined. Current evidence in regard to protein blends × exercise interactions is sparse, however, daily supplementation of a soy and dairy protein blend (containing 25% whey protein, 25% soy protein and 50% casein protein) during whole-body RET for 12 weeks tended to increase lean body mass compared to maltodextrin control, with no trend observed in a whey protein supplemented group [31]. Whilst this may be a reflection of the beneficial divergent digestive properties of these protein sources, further research is required to confirm or refute this.
Although the consumption of animal and plant protein-blends may be suitable for some, they will not be suitable for all (i.e., vegans) and as such, sustainable plant-and plant-derived protein blends (i.e., blending two or more different plant protein sources) are an emerging area of interest and research. Whilst there is no experimental evidence available to date, it is plausible that combining a plant-derived protein source low in lysine and high in methionine (e.g., rice) with another plant-derived protein source with a divergent EAA profile (e.g., pea protein) will provide a plant protein blend that satisfies all of the EAA necessary for robustly stimulating MPS [7,9]. Indeed, researchers have started to develop a variety of mixed plant-protein blends that exceed current AA requirements [9]. However, whether these blends stimulate MPS similarly to animal-derived proteins remains to be seen [9]. Further, the efficacy of plant and collagen protein blends for muscle health (both with and without animal-derived protein) warrants future research, particularly since collagen may support ECM remodelling and thus may be particularly effective in the context of acute/chronic exercise.
In summary, the limited available evidence suggests that animal and plant protein blends may support anabolic responses to acute exercise.

Future Directions
Despite a wealth of protein source research to date, many gaps remain in our understanding of how animal, plant, collagen and blended protein sources can modulate musculoskeletal outcomes, particularly in the context of ageing, exercise and energy restriction. Here, we outline some of the key gaps that we suggest are worthy of imminent future investigation.
Whilst animal-derived protein sources are by far the most investigated protein source to date, the length of time that the muscle remains refractory to dietary animal-derived protein following the onset of "muscle full" remains to be determined. Whether the kinetics of this refractory period (e.g., duration) are dependent on the protein source (i.e., plant, collagen, blended) also remains to be investigated thoroughly. Further, whilst much research has investigated the optimal (e.g., amount, type, etc.) animal-derived protein feeding strategy, the optimal type, texture, matrix and amount of animal protein that is most beneficial for maintaining and potentiating musculoskeletal health (i.e., muscle and bone) during energy restriction remains to be fully investigated.
Similarly, there is a lack of rigorous experimental findings in regard to the effects of plant-derived protein sources on both muscle and bone health during energy restriction, warranting further investigation.
In regard to collagen protein, further research should look to substantiate previous evidence suggesting that collagen protein may support blood flow restriction RET-induced muscle growth. In the context of bone health, collagen protein provided in conjunction with vitamin-C may herald anabolic bone remodelling effects, although this remains to be investigated. Since much less research exists regarding collagen protein (compared with animal-and plant-derived protein), much more work is needed to determine the optimal collagen protein dosing strategy (e.g., amount, timing), including adjuvant nutritional needs (i.e., vitamin-C, protein blends), that is most beneficial for potentiating muscle and bone health, particularly in the context of exercise and energy restriction.
Finally, whilst there is evidence to suggest that protein blends may support musculoskeletal remodelling in response to acute exercise, further evidence is required to clarify the effects in regard to supporting chronic RET-induced musculoskeletal adaptations. Theoretically, mixed plant, plant and collagen, animal and collagen, and animal and plant blends each have anabolic potential, however, the most sustainable and efficacious protein blend for anabolic stimulation in the context of exercise, ageing and energy restriction remains to be determined.

Conclusions
In conclusion, an increased appreciation of the role of protein sources (including protein blends) in relation to the musculoskeletal system under beneficial (e.g., exercise) and deleterious (e.g., ageing, energy restriction) perturbations is crucial to informing appropriate nutritional support in the face of complex challenges, such as changes in appetite and/or socio-economic trends. Plant-derived proteins may provide suitable alternatives to animal proteins in relation to musculoskeletal health, albeit under some circumstances at higher-intakes. Similarly, collagen-derived proteins represent relatively nitrogen-dense sources that have shown some efficacy in relation to favourable body composition changes. Protein blends harnessing the biological benefits of distinct protein sources may represent a means by which to maximise the health benefits of dietary proteins in relation to musculoskeletal health. A schematic representation of this conclusion is presented in Figure 1. animal protein that is most beneficial for maintaining and potentiating musculoskeletal health (i.e., muscle and bone) during energy restriction remains to be fully investigated.
Similarly, there is a lack of rigorous experimental findings in regard to the effects of plantderived protein sources on both muscle and bone health during energy restriction, warranting further investigation.
In regard to collagen protein, further research should look to substantiate previous evidence suggesting that collagen protein may support blood flow restriction RET-induced muscle growth. In the context of bone health, collagen protein provided in conjunction with vitamin-C may herald anabolic bone remodelling effects, although this remains to be investigated. Since much less research exists regarding collagen protein (compared with animal-and plant-derived protein), much more work is needed to determine the optimal collagen protein dosing strategy (e.g., amount, timing), including adjuvant nutritional needs (i.e., vitamin-C, protein blends), that is most beneficial for potentiating muscle and bone health, particularly in the context of exercise and energy restriction.
Finally, whilst there is evidence to suggest that protein blends may support musculoskeletal remodelling in response to acute exercise, further evidence is required to clarify the effects in regard to supporting chronic RET-induced musculoskeletal adaptations. Theoretically, mixed plant, plant and collagen, animal and collagen, and animal and plant blends each have anabolic potential, however, the most sustainable and efficacious protein blend for anabolic stimulation in the context of exercise, ageing and energy restriction remains to be determined.

Conclusions
In conclusion, an increased appreciation of the role of protein sources (including protein blends) in relation to the musculoskeletal system under beneficial (e.g., exercise) and deleterious (e.g., ageing, energy restriction) perturbations is crucial to informing appropriate nutritional support in the face of complex challenges, such as changes in appetite and/or socio-economic trends. Plant-derived proteins may provide suitable alternatives to animal proteins in relation to musculoskeletal health, albeit under some circumstances at higher-intakes. Similarly, collagen-derived proteins represent relatively nitrogen-dense sources that have shown some efficacy in relation to favourable body composition changes. Protein blends harnessing the biological benefits of distinct protein sources may represent a means by which to maximise the health benefits of dietary proteins in relation to musculoskeletal health. A schematic representation of this conclusion is presented in Figure 1.