The main protein expert group for the adult population made the research questions in cooperation with other relevant expert groups including the Elderly Group, see Appendix 1.

We included studies reporting protein intake from foods, but excluded studies using isolated protein as supplements, as well as studies based on the intake of certain amino acids.

The protein expert group that addressed the adult population defined the search terms relevant for both adults and the elderly in collaboration with a librarian, see Appendix 2. The databases used were PubMed and SweMed (the latter was used to identify Nordic papers not published in PubMed). The main search included the period January 2000 to January 2011. An additional search was run in Medline through the PubMed platform in January 2012 in order to update the search with the most recent papers published from January to December 2011.

Screening of all the papers was carried out by the two authors. The first screening included 301 abstracts sent from the main protein group. All articles suggested by at least one of the two experts were ordered as full text papers. The second screening included 152 full text papers, and papers suggested by at least one expert were included in the quality assessment. The quality assessment was done according to the principles in the guidelines (7). Briefly, a quality assessment tool specific for the study type was used to grade the papers as A=high quality study with very low level of potential bias; B=some bias, but not enough to invalidate the results; C=significant bias and weaknesses that may invalidate the results. Then, after the quality grading, evidence tables were constructed with a description and the quality assessment of each study. Finally, for each evaluated outcome with more than one article, the grading of evidence was based on summary tables and a four-class grading: convincing (high), probable (moderate), suggestive (low), and inconclusive (insufficient). The minimum requirement for suggestive was two studies showing an association, and no conflicting results. If some studies showed a non-significant (neither positive nor negative) association, it was decided that for suggestive evidence the number of studies showing an association was required to be at least two higher than those showing no association.

The studies used for the grading of evidence for protein requirements based on N-balance studies are one meta-analysis including 19 N-balance studies (3), quality graded as B, and four controlled metabolic studies (8–11), quality graded as B, A, B, and B, respectively, see Appendix 4, Table 1.

Rand et al.'s meta-analysis from 2003 (3) included 19 N-balance studies of eucaloric diets with at least three test protein intakes given. Only one study (4) in the meta-analysis reported individual data on requirements of older subjects. The meta-analysis found no significant differences in requirements between adult age, sex, or source of dietary protein, but they also stated that the data did not provide sufficient power to detect possible differences. The median estimated nitrogen requirement was 105 mg N/kg BW/day corresponding to 0.66 g good-quality protein/kg BW/day, and based on the 97.5th percentile, the estimated recommended dietary allowance (RDA) was set to 0.83 g good-quality protein/kg BW/day.

In 2001, Campbell et al. (8) conducted a small 14-week strictly controlled metabolic study with 10 healthy elderly men and women who were provided 0.8 g protein/kg BW in a eucaloric diet. There was no young control group. Steady state was reached at week 2 indicating that the protein intake was adequate for the participants. But hereafter the urinary nitrogen excretion decreased and was associated with a loss in mid-thigh muscle area. The authors suggested that the protein intake might have been marginally inadequate and result in longer-term accommodation in skeleton muscle.

In a controlled metabolic study by Morse et al. (10), 11 healthy elderly women were provided diets with three different protein intake levels, that is, low protein diet: 0.5 g/kg BW; medium: 0.75 g/kg BW; high: 1.0 g/kg BW during three 18-day periods with a minimum 1-week habitual diet between the periods. N-balance was determined at week 2 and 3 of each diet. Mean adequate protein allowance was estimated to be 0.90 g/kg BW at week 2 and 0.76 g/kg BW at week 3, but the urinary nitrogen excretion decreased between week 2 and 3 indicating that a steady state was not reached yet.

In 2008, Campbell et al. (9) tested young versus old, and men versus women in a high quality controlled metabolic study with a low protein (0.5 g/kg BW), medium protein (0.75 g/kg BW) and high protein (HP) (1.0 g/kg BW) diet. The included elderly women were apparently the same as in the study by Morse et al. (10). After 18 days, the N-balance was not different between the four groups, and the estimated requirement expressed per kg BW was not significantly different for the young versus old, or men versus women. Mean protein requirement was lower for older women versus older men, but expressed per kg fat-free mass (FFM) there was no significant difference. For all subjects combined, the adequate protein allowance was estimated to be 0.85±0.21 g/kg BW/day, and not statistically different from the estimate of 0.83 g/kg BW/day, as suggested by Rand et al. (3).

A short-term study was also included because of an HP intake in the test meal versus usual protein (UP) intake (11). Young men and women (UP 1.04 g/kg BW and HP 2.08 g/kg BW) versus old men and women (UP 0.89 g/kg BW and HP 1.79 g/kg BW) were tested for 10 days on each diet in a cross-over design. There was no age related difference in N-balance. Still, there was concern about an HP diet corresponding to ca. 24 E% in the elderly because of a potentially negative effect on the kidney function expressed as lack of increase in glomerular filtration rate (GFR) from a habitual low GFR value among the elderly participants.

In summary, the evidence is assessed as probable regarding the estimated average requirement (EAR) of 0.66 g good-quality protein/kg BW per day and the subsequent RDA of 0.83 g good-quality protein/kg BW per day for all adult age groups, including the elderly, based on N-balance studies (Table 1).

The evidence of potential adverse effects of a HP diet (ca. 24 E%) based on only one study is regarded as inconclusive (Table 1).

The evaluation of the association between protein intake and muscle mass among healthy elderly persons is based on one randomized controlled trial (12), quality graded as B, and two prospective cohort studies (13, 14), quality graded as B and C, respectively, see Appendix 4, Table 2.

In a small study with 12 healthy elderly women, Castaneda et al. (12) provided two weight-maintaining diets with marginal protein (0.45 g/kg BW) and adequate protein (0.92 g/kg BW), respectively. This corresponded to an estimated protein E% of ca. 6 E% and 13 E%, respectively. After 10 weeks, the women with the marginal protein intake had statistically significant decreased muscle mass from 17±0.9 to 14.7±0.8 kg, and decreased muscle fiber cross-sectional area (CSA) (type I fibers) as well as a decrease in the protein-sensitive anabolic mediator insulin-like growth factor-1, IGF-1, while the women with the adequate protein intake achieved increased muscle fiber CSA and IGF-1.

In the Health ABC study, Houston et al. (13) measured total lean mass (LM) and non-bone appendicular LM (aLM) with DXA in community-dwelling older men and women. Total, animal and vegetable protein intake were measured with a food frequency questionnaire (FFQ) at baseline and expressed in energy adjusted g/day and E%. The mean daily protein intake was 0.9 g/kg BW, and after 3-year follow-up the mean decrease in LM and aLM was 0.68 ±1.94 and 0.48±1.08 kg, respectively. Those in the highest quintile of protein intake ( ≈19 E%) lost 40% less LM and aLM compared to the lowest quintile ( ≈11 E%). The change in LM and aLm was statistically significant related to total and animal protein intake, but not to vegetable protein intake. However, there was no statistically significant difference in loss of LM between quintiles among the subgroup of weight stable participants (49.5%).

As part of a prospective randomized controlled cohort trial of supplemental calcium to prevent fractures, Meng et al. (14) analyzed the association between total protein intake and body composition after 5 years, expressed as LM and bone mineral content (BMC) in elderly women. They found that those in the highest tertile of total protein intake (T3: >1.6 g/kg BW or 20 E%) compared to the lowest tertile (T1: 0.85 g/kg BW or 18 E%) had higher LM (T3: 37.4±4.8 kg versus T1: 35.5±4.5 kg) and BMC, independent of age, body size, energy intake and physical activity. The energy intake in the lowest tertile of protein intake ( ≈5.3 MJ) indicates underreporting, and thus the actual protein intake is difficult to assess.

The evidence is assessed as suggestive regarding a positive relation between muscle mass and total protein intake in the range of 13–20 E% (Table 2).

The evaluation of the association between protein and bone health is based on two randomized controlled trials, eight prospective cohort studies, and one case–control study, see Appendix 4, Table 3.

The evaluation of the association between protein and physical training is based on only two randomized controlled trials (25, 26), both quality assessed as B, see Appendix 4, Table 4.

In a strictly controlled metabolic study by Campbell et al. (25), all subjects were assured to have a regular protein intake of 0.8 g protein/kg BW per day in euenergetic menus in order to maintain BW stability, whereas they were randomized to resistance training or a sedentary behavior for 12 weeks. The 17 old adults (mean age about 65 years) who trained were able to increase strength and their muscle mid-thigh area (measured with CT), whereas fat-free mass and body water (measured with deuterium dilution) decreased. The sedentary group had a decrease in mid-thigh muscle area by an intake of 0.8 g protein/kg BW ( ≈10 E%).

The study by Haub et al. (26) showed that the effect on strength and muscle mass by resistance training for 12 weeks in 21 men aged 65±5 years during weight stability did not differ with protein source, that is, beef-containing diet or lacto-ovo-vegetarian diet. The diets altogether provided more than 1 g protein/kg BW per day (range 1.03–1.17 g/kg BW). Both groups had increased muscle mass and strength.

Due to few studies, the evidence is assessed as inconclusive regarding the relation of total protein intake and sources of protein (animal versus vegetable protein) to muscle mass and body composition in combination with resistance training.

For some outcomes, only one study was identified on the association to protein intake and thus, the evidence is assessed as inconclusive; see Appendix 3, Table 5.

N-balance remains the method of choice for determining protein requirement in adults. Nevertheless, there are limitations in this method that need to be addressed, such as being related to the accuracy of the measurements, the short duration of the studies and the difficulty in interpretation of the results. Rand et al.'s meta-analysis (3) included 27 N-balance studies with six studies performed in persons aged 63 and older. From those, Rand et al. selected only studies that provided individual data for persons studied at three or more levels of protein intake. Thus, Rand's estimation was based on 19 N-balance studies with 221 ‘young’ individuals and only 14 ‘old’ individuals from a study by Uauy et al. (4). Rand et al. found no statistically significant differences between ‘young’ and ‘old’, sex or source of dietary protein, but the data did not provide sufficient power to detect possible differences. The median nitrogen requirement was 130.5 mg per kg BW per day in the old versus 103.9 mg per kg BW per day in the younger group corresponding to a difference of 0.17 g protein per kg BW per day or a 26% higher requirement. The conclusion that Rand et al. draw from their analysis was that the healthy elderly may have a somewhat higher requirement, but that there was not enough evidence to give age-specific recommendations (3).

The objective of the high quality graded N-balance study by Campbell et al. (9) was to study the effect of age on the EAR. They found no difference in the EAR between the young and old participants, and the calculated adequate protein allowance of 0.85 g good-quality protein per kg BW per day for all participants combined was not statistically different from the RDA estimated in Rand's meta-analysis (3).

An earlier study by Campbell et al. (8) also found 0.8 g protein/kg BW to be sufficient to obtain N-balance among 10 elderly people. A short-term study (11) with only 10 days on each diet was also included in the current systematic review because a HP (22–24 E%) intake was tested against a usual protein (11–12 E%) intake in young as well as elderly healthy participants. The net daily N-balance increased equally in the young and older participants on the HP diet.

From these studies, we assess the evidence as probable regarding the estimated EAR of 0.65 g good-quality protein/kg BW/day and the resulting RDA of 0.83 g good-quality protein/kg BW/day as adequate to meet the minimum dietary protein needs of virtually all healthy elderly persons.

WHO/FAO/UNU (5) based their protein requirement for elderly people on Rand et al.'s meta-analysis (3) and also on a reassessment of the N-balance studies among the elderly who were not included in the meta-analysis and, supported by additional studies with measurements of metabolic demand, WHO/FAO/UNU concluded, that ‘these data lend confidence to the overall conclusion drawn from the nitrogen balance data that the physiological protein requirement does not increase with age’. Based on Rand et al. (3), Campbell et al. (8), and studies before 2000 including some short-term studies, EFSA (6) concluded that ‘the available data are insufficient to specifically determine the protein requirement in older adults and that at least the same level of protein intake as for young adults is required for older adults. As sedentary older adults have a lower energy requirement the protein to energy ratio of this subgroup is higher than for younger adults’.

Long-term health and functional outcomes related to various protein intakes are likely of greater potential importance than N-balance studies that only give short-term indications on what intake is needed to prevent loss of protein mass, that is, mainly muscle mass, and bone mass. It has to be emphasized that the processes of dietary-based losses of muscle mass and strength, that is, sarcopenia, and bone, that is, osteoporosis, are extremely slow. This means that it may take many years before these losses are: (1) possible to identify with current measurement techniques (e.g. DXA); and (2) lead to compromised function, morbidity or mortality. Moreover, there is no strong consensus on what are validated or accepted outcome variables, as well as which are the most reliable biological marker of protein status.

Prevention of sarcopenia, that is, the age-related loss of muscle mass, strength and function is highly relevant. Advanced sarcopenia is a part of physical frailty and thus, associated with increased likelihood of falls and impairment in the ability to perform activities of daily living (33). The protein intake in the prevention of sarcopenia is one of many possible intervention strategies in question.

The studies included in this systematic review also point at a caveat whether the allowance represents a sufficient intake level in relation to loss of muscle mass. Campbell et al. (8) distinguish between adaption and accommodation. Adaptation refers to metabolic changes that occur in response to changes in protein intake and result in the establishment of a new steady state without a compromise or loss in physiological function, whereas accommodation refers to metabolic changes due to the decreased protein intake to establish a new steady state but at the cost of compromised physiological functions. During the small 14-week study, Campbell et al. found that urinary nitrogen excretion decreased after week 2, and the change was associated with a loss of skeletal muscle (mid-thigh muscle area) which points at a longer-term accommodation in skeletal muscle. Additionally, Morse et al. (10) found a decrease in nitrogen excretion between week 2 and week 3 in an 18-day study, but they found no changes in fat-free mass.

Based on a small randomized controlled trial (12) and two prospective cohort studies (13, 14) with 3–5 years follow-up, we assessed the evidence of a relation between prevention of loss of muscle mass and a total protein intake in the range of 13–20 E% to be suggestive. We excluded one study (34) with ‘borderline’ elderly (mean age 62±7 years) according to our eligibility criteria, but the inclusion of this study would not have changed our assessment of evidence. In a strictly controlled metabolic study with focus on N-balance (8) and on resistance training (25), Campbell et al. found that a protein intake of 0.8 g /kg BW ( ≈10 E%) during 14 weeks resulted in a loss of mid-thigh muscle area in the sedentary control group during BW stability. In the Health ABC Study (13), that is the first longitudinal study to examine the role of dietary protein on changes in body composition by using state-of-art-body-composition measurements, the mean protein intake was 0.9 g/kg BW and the mean 3-year loss of lean body mass was 0.68±1.9 kg. Subjects in the highest quintile of protein intake (≈19 E%) lost less LM compared to those in the lowest quintile ( ≈11 E%). But of notice is that there was no statistically significant association between total protein intake and a 3-year loss of muscle mass adjusted for physical activity in the 49.5% of participants that were weight stable, which points to the necessity of a sufficient energy intake among the elderly to keep up the protein stores.

The EFSA Panel (6) considered that in healthy adults the available data on the effects of dietary intake on muscle mass and function did not provide evidence to be considered as a criterion for setting a population reference intake (PRI) and further they stated that ‘There are no data showing that an additional intake of protein would increase muscle mass in different age groups who are in nitrogen balance, including subjects undertaking endurance or resistance exercise’.

During aging there is an average loss of both muscle and bone mass in the population, however, with very wide variations between individuals.

The role of dietary protein on bone health has been controversial. On the one hand, urinary calcium loss is increased by HP intakes, while on the other hand protein increases calcium absorption or bioavailability, which questions the net effect of HP diets on calcium economy and the effect on bone health (35). Any negative effect of protein may also be opposed by an increase in the protein sensitive anabolic mediator insulin-like growth factor 1, IGF-1.

We assess the evidence as suggestive for an association between protein intake and BMD. The evidence regarding the relation of protein to an overall effect on bone health based on studies with bone loss, fracture risk and falls as outcomes are according to our view point inconclusive. Thus, the conservative interpretation is in line with EFSA (6) that found the available evidence regarding protein and bone health to be insufficient.

The positive association between protein intake and BMD/BMC was based on one intervention study (15), and three prospective cohort studies (14, 16) (17), but even though the effect was observed with protein intakes above approximately 0.85 g/kg BW or 16–18 E%, the actual protein intake level was unknown due to underreporting. In the 5-year cohort study by Meng et al. (14), where lean body mass as well as BMC was measured they found that the association between protein intake and BMC disappeared after further adjustment for LM. Thus, the authors concluded that the protein effect on bone mass may be partly mediated by its effect on muscle. In the randomized controlled study with calcium and vitamin D supplementation by Dawson-Hughes and Harris (18), the highest tertile of protein intake (20 E% or 1.2 g/kg BW) was associated with less bone loss compared to the lowest tertile (14 E% or 1.1 g/kg BW), but only in the intervention group. Interestingly, the habitual mean intake in the placebo group was 871 mg calcium and approximately 7-µg vitamin D, which closely corresponds to the NNR from 2004 (1). Thus, the possible effect of protein intake on bone health may depend on an intake level of calcium and vitamin D above the current recommendations.

A prospective cohort study (22) and a case–control study (23) found total protein intake associated with a decreased risk of fractures, but in the case–control study it was only statistically significant among 50–69-year-old participants, while the association was non-significant among the 70–79-year-old participants (23). In the Study of Osteoporotic Fractures in postmenopausal women (21), a high A/V ratio was associated with an increased risk of fractures.

Overall, the animal protein intake showed both positive (17, 19) (23) and negative (21) effects on bone health, while no detrimental effect of total protein intake was observed. No upper limit could be detected for total protein intake in relation to bone health. The study by Hannan et al. (19) even found that elderly with total protein intake up to three times RDA (1.2–2.8 g/kg BW or 18–27 E%) had the least bone loss after controlling for known confounders, including weight changes.

Only few studies address the issue of regular protein intake for optimal effect of physical exercise in healthy old adults. We included two American medium term RCTs (25, 26) that evaluate if the amount or the quality of the protein intake have different effects on muscle mass gain or strength by ∼3 months of resistance training. Due to the few studies, we assess the evidence to be inconclusive.

In addition, there are studies that use supplementation of protein hydrolysates in order to increase the protein intake during periods of resistance training. Such studies fall outside the scope of this review of effects by regular food, but they still provide understanding of potential effects of combined training and increased protein intake, and are thus mentioned briefly. A recent meta-analysis by Cermak et al. from 2012 (36) combined the results from six studies performed in ‘older’ subjects, and it was concluded that protein supplementation – provided either by supplement or via the habitual diet corresponding to 42±30 g – increased the effects on muscle mass by an additional 38% as well as a 33% greater increase in muscle strength (1-RM leg press strength). It should be observed that the authors set the distinction between young and old at 50 years of age. In all but one study participant, average age was below 65 years. However, Verdijk et al. (37) evaluated the effect of 20 g extra protein given three times/week in conjunction with exercise during 12 weeks in 26 healthy men (average age 72±2 years). No further effect on muscle mass or strength of the protein supplementation was observed. Neither was an effect on total protein intake by the supplementation observed, that is, intake being constant at 1.1 g/kg/day in both groups. Interestingly, the separate studies in the meta-analysis by Cermak et al. could not themselves establish evidence for positive effects by supplementation, mainly due to underpowered study designs.

There are two short-term studies that also should be mentioned regarding timing. In the study by Andrews et al. (38), fifty 60–69 year-old subjects were provided a post-exercise drink with 0.4 g protein/kg/day during 12 weeks leading to a range of daily protein intake from 0.72 to 1.35 g/kg. Variability in mean daily protein intake was not associated with a change in LM. In contrast to these results, Esmarck et al. (39) reported that a protein supplementation of 10 g given directly in connection with the training session had positive effects on muscle hypertrophy, mean fiber area and strength, whereas a corresponding intake 2 h after the training session could not provide these effects.

Some outcomes, that is, hypertension, frailty, and mortality, were only addressed in one article, and thus, the evidence was assessed as inconclusive.

Potential risks, e.g. kidney damage or hypertension, of HP intakes need to be addressed. The N-balance study by Walrand et al. (11) found that baseline GFR was lower in the old participants compared to the young participants. The study reported that a HP intake corresponding to 24 E% did not increase GFR in the old participants. This is probably due to the reduced kidney function in elderly, since patients with mild-to-moderate chronic kidney disease also do not show the usual protein-induced increase in GFR (40). The increase in GFR is a normal physiological adaption to increased protein intake (41) but it is also an important component of the hyperfiltration theory of Brenner (42) due to its presumed effect of increasing glomerular pressure. It is also an important component of the hyperfiltration theory that protein overloading of remnant nephron mass should be avoided.

In a prospective cohort study (27), it was reported that animal protein intake increased the risk of hypertension only among persons ≥ 70 years of age after 6 years follow-up.

Except from the strictly controlled metabolic studies it has generally been difficult to assess the actual protein intake in the observational studies due to misreporting (underreporting). The FFQ is a widespread dietary assessment method in observational studies. Based on a thorough calibration study, Beasley et al. (28) concluded that FFQ better assess nutrient consumption as a fraction of total energy intake than absolute nutrient consumption. Thus, it may be more appropriate to conclude from protein E% than protein intake expressed in g per day or per kg BW.

Still, taken together studies on protein intake and long-term outcomes related to muscle mass (8, 12–14, 25), bone mass (14–16, 18), and frailty (28) altogether indicate that there are suggestive evidence for positive effects of a higher intake than the 0.83 g/kg BW/day recommendations based on N-balance studies only.