E ects of Leucine-Enriched Whey Protein Supplementation on Physical Function in Post-Hospitalized Older Adults Participating in 12-Weeks of Resistance Training Program: A Randomized Controlled Trial

nutrients Article Effects of Leucine-Enriched Whey Protein Supplementation on Physical Function in Post-Hospitalized Older Adults Participating in 12-Weeks of Resistance Training Program: A Randomized Controlled Trial Maria Amasene 1, Ariadna Besga 2,*, Iñaki Echeverria 3, Miriam Urquiza 3, Jonatan R. Ruiz 4, Ana Rodriguez-Larrad 3, Mikel Aldamiz 2, Pilar Anaut 2, Jon Irazusta 3and Idoia Labayen 5 1Department of Pharmacy and Food Science, University of the Basque Country UPV/EHU, 01006 Vitoria-Gasteiz, Spain; [email protected] 2Department of Medicine, Araba University Hospital, Bioaraba Research Institute, OSI Araba. CIBERSAM, University of the Basque Country (UPV/EHU), 01004 Vitoria-Gasteiz, Spain; [email protected] (M.A.); mariapilar[email protected] (P.A.) 3Department of Physiology, University of the Basque Country, UPV/EHU, 48940 Leioa, Spain; [email protected] (I.E.); [email protected] (M.U.); ana.r[email protected] (A.R.-L.); [email protected] (J.I.) 4PROFITH “PROmoting FITness and Health through physical activity” Research Group, Sport and Health University Research Institute (iMUDS), Department of Physical Education and Sport, Faculty of Sport Sciences, University of Granada, 18071 Granada, Spain; [email protected] 5ELIKOS group, Institute for Innovation and Sustainable Development in Food Chain (IS-FOOD), Public University of Navarra, 31006 Pamplona, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-680-471-077 Received: 16 September 2019; Accepted: 25 September 2019; Published: 1 October 2019   Abstract: Age-related strength and muscle mass loss is further increased after acute periods of inactivity. To avoid this, resistance training has been proposed as an effective countermeasure, but the additional effect of a protein supplement is not so clear. The aim of this study was to examine the effect of a whey protein supplement enriched with leucine after resistance training on muscle mass and strength gains in a post-hospitalized elderly population. A total of 28 participants were included and allocated to either protein supplementation or placebo supplementation following resistance training for 12 weeks (2 days/week). Physical function (lower and upper body strength, aerobic capacity and the Short Physical Performance Battery (SPPB) test), mini nutritional assessment (MNA) and body composition (Dual X-ray Absorptiometry) were assessed at baseline and after 12 weeks of resistance training. Both groups showed improvements in physical function after the intervention (p<0.01), but there were no further effects for the protein group (p>0.05). Muscle mass did not improve after resistance training in either group (p>0.05). In conclusion, 12 weeks of resistance training are enough to improve physical function in a post-hospitalized elderly population with no further benefits for the protein-supplemented group. Keywords: elderly; aging; muscle mass; strength; resistance training; leucine; whey protein; protein supplementation 1. Introduction Aging is characterized by a progressive decline in skeletal muscle mass and function defined as sarcopenia [ 1 ]. Sarcopenia is related to an increased risk of falling [ 1 ], fractures [ 2 ], physical Nutrients 2019,11, 2337; doi:10.3390/nu11102337 www.mdpi.com/journal/nutrients Nutrients 2019,11, 2337 2 of 15 disability [ 3 ] and mortality [ 4 ]. In healthy aging, muscle mass loss ranges from 3% to 8% per decade [ 5 ]. However, this decline is further emphasized by acute or chronic illness [ 6 ], inactivity [ 6 ] and inadequate protein and/or energy intake [ 7 ]. So, physical activity is proposed as an effective countermeasure to delay the age-related muscle mass loss [ 7 ]. Indeed, following a healthy lifestyle may help to prevent and reduce the consequences of age-related muscle mass loss [7]. A balanced protein metabolism is important to muscle mass accretion and maintenance [ 8 ]. Energy and protein intake are key nutritional factors to achieve protein balance [ 7 ]. However, due to several physiological and social factors, elderly people tend to reduce food intake and, in consequence, often fail to meet energy and protein requirements [ 9 ]. Likewise, protein-energy malnutrition is frequent in elderly patients [ 9 ]. Besides total daily protein intake [ 10 ], dietary protein quality and its anabolic potential have also received increased interest with the goal of optimizing skeletal muscle anabolism in the elderly [10,11]. Dietary protein quality depends on its digestibility, amino acid (AA) profile and AA availability [12,13] . Therefore, in studies aiming to optimize muscle mass among elderly people, whey protein ( ≈ 20 g/day) is considered superior to other isolated protein sources [ 14 , 15 ]. Whey protein is also characterized for being a high leucine-containing protein, which is the main precursor for activating muscle protein synthesis via mammalian target of rapamycin (mTOR) signaling [ 8 , 16 ]. A protein/AA source containing around 1.8–2.0 g of leucine would be enough to activate post-exercise “leucine trigger”, whereas in rested conditions, a higher dose might be required in young adults [ 16 ]. Other authors [ 15 ], reported that 20 g of whey protein enriched with 3 g of leucine post-exercise resulted in a greater muscle protein synthesis rate in healthy older people. Muscle mass accretion and strength gain depend on the synergistic effect of protein consumption and resistance training [ 8 , 16 ]. Protein ingestion close after exercise seems to increase exercise-induced muscle mass sensitivity to anabolism [ 8 ]. In a recent systematic review and meta-analysis [ 17 ], it was concluded that a combination of protein supplementation and resistance training led to positive effects on body composition, muscle volume and strength, and physical function in elderly people. In contrast, in people aged 70 years or older, it was shown that despite overall improvements from baseline for the majority of outcomes, there were no significant differences between the group receiving protein/AA supplementation along with resistance training and the group with resistance training alone [18]. Acute periods of inactivity, such as a hospital stay, accentuates age-related muscle mass loss [ 6 ]. After hospitalization, older individuals are more vulnerable to develop any adverse event [ 19 ]. Then, early interventions to accelerate recovery and avoid hospital readmission will be important [ 20 ]. For example, implementing interventions that combine nutrition and physical exercise immediately after discharge [20]. In view of this growing interest, the objective of the present study was to examine the effect of a whey protein supplement enriched with leucine after resistance training on muscle mass and strength gains in a post-hospitalized elderly population. We hypothesized that elderly people after hospitalization may benefit most from the synergetic effect of protein supplementation and a resistance training session. 2. Methods 2.1. Study Design The Sarcopenia and Fragilidad-protein (S and F-PROT) study is a prospective, 24-week, single-blind, randomized, placebo-controlled clinical trial (ClinicalTrials.gov ID: NCT03815201). The study was conducted at the facilities of the Araba University Hospital in Vitoria-Gasteiz (North Spain), from September 2017 to July 2018. The Clinical Research Ethics Committee of the Araba University Hospital (CEIC-HUA: 2017-021) approved the study protocol (S and F-PROT) that complied with the revised ethical guidelines of the Declaration of Helsinki (revision of 2013). All participants Nutrients 2019,11, 2337 3 of 15 were informed about the details of the research and signed an informed consent before their enrolment in the study. The S and F-PROT project compared relative changes on functional capacity (muscular strength of upper and lower limbs, and aerobic capacity), body composition (lean mass and fat mass at whole body, arms, legs and trunk) and nutritional status between two groups following a resistance training intervention program with post-exercise supplementation (Protein-group) or without supplementation (Placebo-group). 2.2. Participants Volunteers accessed the program after hospitalization at the internal medicine service of the Araba University Hospital, or by medical recommendation at the outpatient internal medicine specialty at the Araba University Hospital (Figure 1). Hospitalized patients older than 70 years old were first pre-screened for eligibility. All pre-screened participants met the following criteria: >70 years old, a punctuation of ≥ 20 at the Mini Mental State Questionnaire (MMSE), fulfilled the criteria for sarcopenia diagnosis of the European Working Group on Sarcopenia in Older People, were able to walk alone or using a walking stick, a walking frame, or parallel walking bars, were able to understand the instructions or what had being said, and signed the informed consent. Patients were excluded for examination if they had any of the following exclusion criteria: history of chronic kidney disease, had suffered a heart attack in the last 3 months, been unable to walk, have suffered any fracture of the upper or lower limbs in the last 3 months, been suffering from severe dementia, a history of autoimmune neuromuscular disorders (for example, myasthenia gravis, Guillain–Barr é syndrome, inflammatory myopathies) or amyotrophic lateral sclerosis, or refused to sign the informed consent. Patients that were eligible for the intervention program were assessed for nutritional status (Mini Nutritional Assessment-Short Form (MNA-SF; Nestl é Nutrition Institute)) [ 21 ], physical function (Short Physical Performance Battery (SPPB) [ 22 ] and handgrip strength), frailty (a Spanish language version of the Fried test [ 23 ]) and cognitive function (Spanish validated version of the Pfeiffer test, the Short Portable Mental Status Questionnaire (SPMSQ) [ 24 ]) during their hospitalization. Patients were informed about the possibility of participating in an exercise training program after hospital discharge and an informed consent was given along with further written information. After a recovery week, patients were cited for baseline physical function assessment before initiating the intervention program. Many hospitalized patients did not meet inclusion criteria when assessing eligibility (21.8%) or refused to participate (66.6%) because of health issues, lack of interest in the physical exercise program, or had problems to get to hospital for the intervention sessions. As the hospital recruitment proved not to be enough for the intervention aims, the outpatient internal medicine service was chosen as an alternative recruitment source. Those patients at the outpatient internal medicine service potentially meeting inclusion criteria were informed by their doctor about the exercise intervention program. Thereafter, patients were cited for a first eligibility assessment with the investigation team. If participation criteria were met, patients were again cited a week after for baseline physical function assessment (Figure 1). Nutrients 2019,11, 2337 4 of 15 Nutrients 2018, 10, x FOR PEER REVIEW 4 of 16 Figure 1. Flow Diagram of participants. Patients that were eligible for the intervention program were assessed for nutritional status (Mini Nutritional Assessment-Short Form (MNA-SF; Nestlé Nutrition Institute)) [21], physical function (Short Physical Performance Battery (SPPB) [22] and handgrip strength), frailty (a Spanish language version of the Fried test [23]) and cognitive function (Spanish validated version of the Pfeiffer test, the Short Portable Mental Status Questionnaire (SPMSQ) [24]) during their hospitalization. Patients were informed about the possibility of participating in an exercise training program after hospital discharge and an informed consent was given along with further written information. After a recovery week, patients were cited for baseline physical function assessment before initiating the intervention program. Many hospitalized patients did not meet inclusion criteria when assessing eligibility (21.8%) or refused to participate (66.6%) because of health issues, lack of interest in the physical exercise program, or had problems to get to hospital for the intervention sessions. As the hospital recruitment proved not to be enough for the intervention aims, the outpatient internal medicine service was chosen as an alternative recruitment source. Those patients at the outpatient internal medicine service potentially meeting inclusion criteria were informed by their doctor about the exercise intervention program. Thereafter, patients were cited for a first eligibility assessment with the investigation team. Figure 1. Flow Diagram of participants. 2.3. Randomization Following baseline physical function assessment, participants were randomly allocated to one of the two intervention groups: Placebo-group or Protein-group. Participant stratification was based on gender to ensure equal allocation in both groups. 2.4. Supplementation and Blinding Placebo and protein supplements were delivered by the nutritionist in the first half hour following each training session. The protein supplement contained 20 g of whey protein isolate (Davisco ® : BiPRO all-natural whey protein isolate, Eden Paririe, MN, USA) enriched with 3 g of leucine (Nutricia, Madrid, Spain). The nutritional composition of both the placebo and protein supplement is shown in Table 1. The supplements were energy-matched and were flavored with lemon flavor and solubilized in 150 mL of water. Only participants were blinded for supplementation. Supplements were stored in boxes and only the research team could identify them. All supplements were developed, prepared and stored in boxes by Laboratorium Sanitatis SL (Tecnalia Research and Innovation, Vitoria-Gasteiz, Spain). Nutrients 2019,11, 2337 5 of 15 Table 1. Nutritional composition of the protein and placebo supplements. Nutritional Composition Protein Supplement B-lactoglobulin (g/bottle) 20 L-Leucine (g/bottle) 3 Sodium saccharin (g/bottle) 0.050 Sucralose (g/bottle) 0.030 Lemon flavor 654500 (g/bottle) 0.250 Placebo supplement Maltodextrin (g/bottle) 23 Hydroxyethylcellulose (g/bottle) 0.200 Lemon flavor 654500 (g/bottle) 0.250 2.5. Design of the Resistance Training Program Both groups followed a supervised resistance training program for 12 weeks. The program consisted of 1 h sessions on two non-consecutive days per week. The first week of intervention was used for familiarization, and 1-RM (repetition maximum) estimation by the individual’s functional capacity through Brzycki equation [ 25 ]. The load was then gradually increased during a month, and half exercises were performed at 50%–65% of the estimated 1-RM. During the subsequent months, load was increased until 70% of the estimated 1-RM was reached. Two sets were performed per exercise and load and maximum repetition for each exercise was personalized for each participant. All resistance training sessions were designed and supervised by a sport scientist with experience in resistance training for the elderly. All training sessions started with warm-up exercises (heel stand, calf raises, chair stand exercise and neck movements) and were followed by strengthening exercises of upper and lower limbs (arm-curl exercise with the participant in a seated position and personalized load, knee extension exercise with personalized load in a seated position, standing knee flexion with personalized load, side hip raise, standing hip extension and chair stand exercise). In the same resistance training session, some exercises for dynamic balance improvement were also practiced (side-by-side stand, semi-tandem stand, tandem stand, monopodal stand, timed up and go, stepping around obstacles and step up and down exercises). The session finished with 5 min of cool-down, consisting mainly of stretching exercises. 2.6. Outcome Measures Primary and secondary outcomes were assessed at baseline and after 12 weeks of intervention by the same trained researchers. Post-intervention measurements were scheduled within one week following the last exercise session. 2.7. Primary Outcome: Physical Function Physical function was assessed at baseline and at week 13 (once supervised intervention period was finished). Physical function was assessed using a combination of tests. The tests used to assess lower and upper body strength and aerobic capacity were based on the Senior Fitness Test [ 26 ]. For lower and upper body strength, 30-Second Chair Stand Test and 30-Second Arm Curl Test were used, respectively. For upperbody strength, isometrichandgrip strength was alsomeasuredusing ahandled dynamometer (JAMAR ® PLUS +Hand dynamometer). Aerobic capacity was assessed by the 6 min walking test (6MWT). Hence, for physical function assessment, the SPPB test battery was also used [ 22 ]. This test includes the 4 m walking speed test, the standing balance test (side-by-side stand, semi-tandem stand and tandem stand) and the time to rise from a chair five times test [22]. Nutrients 2019,11, 2337 6 of 15 2.8. Secondary Outcomes 2.8.1. Nutritional Assessment A nutritionist completed all nutritional questionnaires along with the participant and/or participant’s relative or caregiver. Participant’s nutritional status was assessed using the MNA questionnaire (Nestl é Nutritional Institute) [ 27 ]. This questionnaire contains 18 items divided into 4 categories: anthropometric assessment, general assessment, short dietary assessment and subjective assessment [ 27 ]. Each answer has a numerical value contributing to the final punctuation. A maximum of 30 points can be obtained. Punctuation ranging from 24 to 30 reflects normal nutritional status, from 17 to 23.5 risk of malnutrition and a punctuation under 17 reflects malnutrition [27]. 2.8.2. Body Composition Body fat, lean mass, bone mass, bone mineral density (BMD) and bone mineral content (BMC) were assessed by dual-energy X-ray absorptiometry (DXA; HOLOGIC, QDR 4500). Body mass (OMRON HN-288, Digital Personal Scale, Barcelona, Spain) was measured barefoot following the standard protocols. Height was estimated using knee height determination (SECA 220, Hamburg, Germany) [ 28 ]. Body mass index (BMI) was calculated as body weight divided by height squared (kg/m 2 ). Waist circumference, hip circumference, calf circumference and mid-arm circumference were measured with a nonelastic tape (CESCORF, Porto Alegre, Brasil) following the protocol recommended by the International Society for the Advancement of Kinanthropometry (ISAK). 2.8.3. Biochemical Parameters Biochemical parameters were obtained from fasting venous blood samples in Ethylenediaminetetraacetic acid -containing tubes and in serum tubes. These tubes were immediately carried to the laboratory and EDTA-containing tubes were centrifuged at 1000 × g at 4 ◦ C for 10 min, whereas serum tubes were centrifuged 90 min after blood collection at 1000 × g at 20 ◦ C for 15 min. Serum albumin, prealbumin and creatinine were measured as protein malnutrition markers. 2.9. Statistical Analysis Baseline characteristics between groups (i.e., placebo versus protein supplementation) were compared using independent Student’s t-test. Sample size estimation and power analysis was calculated for muscle mass increase. With a population size of 35 on each group, a significant alpha level of 0.05, and power >80%, the range for a statistically detectable change in muscle mass will be 1.5–2kg with a standard deviation of 1.5–1.7 kg. Data analysis was performed following the per-protocol principle. Changes in primary and secondary outcomes were calculated as Post-intervention minus Pre-intervention values. Differences between the placebo and the protein groups (fixed factor) in changes on primary outcomes and secondary outcomes were calculated by analyses of covariance adjusting with baseline values. All statistical analyses were performed using the statistical software SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) with a level of significance of α =0.05. Data are expressed as means ± standard error of the mean. 3. Results During the recruitment period, a total of 476 hospitalized patients were evaluated and invited to participate in the study. From them, only 74 (15.5%) accepted to participate, whereas the remaining 402 patients refused to participate (83.4%) or did not meet inclusion criteria (1.15%). Finally, from the 74 patients who accepted to participate, a total of 29 participants (39.2%) were randomized for the study. Overall, 93.9% of the evaluated hospitalized patients did not participate in the study due to inclusion criteria or rejection to participate in the program. Regarding the recruitment from the outpatient internal Nutrients 2019,11, 2337 7 of 15 medicine service, a total of 22 patients were recommended to participate, 40.9% of these patients refused to enter the intervention program or did not meet inclusion criteria (4.5%), whereas 54.54% accepted. In total, 41 patients were randomized for the intervention program, 20 entered the Placebo-group and 21 the Protein-group. From the allocated participants, 13 did not complete the 12 weeks of the intervention program (7 from the Placebo-group and 6 from the Protein-group). The main reason for dropping out from the study in both groups was that participants refused to continue in the program (15.0% of the randomized patients in the Placebo-group and 14.3% in the Protein-group). In the Protein-group, an adverse event was reported with protein supplementation regarding itchy throat and difficulties to inhale, whereas another participant refused to take the protein supplement, so both participants were dropped from the study (Figure 1). Baseline characteristics of the recruited participants can be found in Supplemental Table S1. Table 2shows baseline characteristics of participants. There were no statistically significant differences in body composition and nutritional status variables between groups at baseline. However, within the physical function parameters, the protein-group walked significantly more meters in the 6MWT at baseline (p<0.05). In contrast, the Protein-group showed significantly greater lean mass on the legs (%) than the Placebo-group (p<0.05). Table 2. Characteristics of participants completing the study (intend-to-treat analyses). N Placebo Group N Protein Group p Age (years) 13 81.7 (6.45) 15 82.9 (5.59) 0.607 Women (N, %) 13 7 (53.8) 15 7 (46.7) 0.717 Body mass (kg) 13 75.9 (17.95) 15 68.0 (11.43) 0.188 BMI (Kg/m2)13 30.8 (6.53) 15 27.4 (3.50) 0.110 Physical Function Handgrip (kg/body mass) 13 0.3 (0.09) 15 0.4 (0.09) 0.063 SFT chair stand test 30sec 13 10.6 (4.17) 15 12.3 (2.97) 0.229 SFT arm curl test 30sec 13 13.5 (5.22) 15 16.3 (3.92) 0.137 SFT 6MWT (m) 13 314.8 (139.36) 15 411.5 (80.40) 0.040 SPPB total punctuation 13 8.7 (2.36) 15 10.1 (1.58) 0.089 SPPB 5Squat 13 14.7 (6.85) 15 12.2 (2.86) 0.232 Body composition Waist to hip ratio 13 1.00 (0.07) 15 0.98 (0.09) 0.459 Lean mass arms (kg) 13 2.3 (0.67) 15 2.3 (0.44) 0.897 Lean mass legs (kg) 13 6.8 (1.70) 15 6.4 (1.08) 0.441 Lean mass trunk (kg) 13 23.0 (4.83) 15 21.5 (3.89) 0.380 Total lean mass (kg) 13 45.2 (9.85) 15 42.3 (6.63) 0.391 Fat mass arms (%) 13 2.6 (0.96) 15 2.4 (0.77) 0.545 Fat mass legs (%) 13 5.8 (1.85) 15 5.4 (1.84) 0.603 Fat mass trunk (%) 13 17.1 (3.87) 15 14.9 (3.03) 0.124 Total fat mass (%) 13 35.4 (8.05) 15 32.1 (6.84) 0.259 Nutritional Status MNA score 13 23.1 (3.82) 15 24.5 (2.11) 0.273 Normal nutritional status (N, %) 13 4 (30.8) 15 11 (73.3) 0.064 At risk of malnutrition (N, %) 13 8 (61.5) 15 4 (26.7) Malnourished (N, %) 13 1 (7.7) 15 0 (0) Biomarkers Creatinine (mg/dL) 10 1.1 (0.48) 15 0.9 (0.35) 0.401 Albumin (g/dL) 13 4.0 (0.39) 15 4.0 (0.31) 0.994 Prealbumin (mg/dL) 12 22.2 (6.63) 14 23.3 (4.31) 0.613 BMI: body mass index; MNA score: Mini Nutritional Assessment score; SFT chair stand test 30 s: Senior Fitness Test chair stand test 30 s; SFT arm curl test 30 s: Senior Fitness Test arm curl test 30 s; SFT 6MWT (m): Senior Fitness Test 6-min Walking Test (m); SPPB total punctuation: Short Physical Performance Battery total punctuation; SPPB 5Squat: Short Physical Performance Battery 5Squat. Values are means and standard deviations. 3.1. Effects of the Intervention on Primary Outcomes: Physical Function Both groups showed improvements over time in all the physical function tests (p<0.01), except for the handgrip strength test (Table 3). However, we did not observe any significant difference between groups in any of the measured physical function tests (Table 3). Nutrients 2019,11, 2337 8 of 15 Table 3. Body composition, nutritional status and physical function in elderly patients before (Pre) and after (Post) their participation in the resistance exercise intervention program plus protein supplementation (Protein-group) or placebo (placebo-group) (analyses per protocol). Placebo-Group Protein-Group Differences between Groups N Pre Post pN Pre Post p∆Placebo ∆Protein p Primary outcome Physical function Handgrip (kg/body mass) 13 0.3 (0.09) 0.3 (0.09) 0.775 15 0.4 (0.09) 0.4 (0.09) 0.651 0.0 (0.03) -0.0 (0.06) 0.971 SFT chair stand test 30sec 13 10.6 (4.17) 13.5 (4.59) 0.003 15 12.3 (2.97) 14.4 (3.22) <0.001 2.8 (2.79) 2.1 (1.53) 0.480 SFT arm curl test 30sec 13 13.5 (5.22) 21.9 (4.66) <0.001 15 16.3 (3.92) 23.5 (4.53) <0.001 8.4 (5.74) 7.2 (4.86) 0.724 SFT 6min WT (m) 13 314.8 (139.36) 375.0 (128.39) 0.002 15 411.5 (80.4) 455.1 (81.77) 0.005 60.2 (53.67) 43.6 (51.2) 0.959 SPPB total score 13 8.7 (2.36) 10.3 (1.89) 0.001 15 10.1 (1.58) 11.3 (0.96) 0.002 1.6 (1.39) 1.2 (1.21) 0.634 SPPB 5Squat 13 14.7 (6.85) 10.6 (3.67) 0.005 15 12.2 (2.86) 10.0 (2.81) 0.004 −4.1 (4.32) –2.2 (2.4) 0.491 Secondary outcomes Body composition Body mass (kg) 13 75.9 (17.95) 75.6 (18.31) 0.621 15 68.0 (11.43) 68.3 11.07) 0.500 −0.3 (2.24) 0.3 (1.60) 0.471 BMI (kg/m2)13 30.8 (6.54) 30.7 (6.64) 0.575 15 27.4 (3.5) 27.5 (3.37) 0.453 −0.3 (2.24) 0.3 (1.60) 0.493 Waist to hip ratio 13 1.00 (0.07) 1.00 (0.08) 0.818 15 0.98 (0.09) 0.96 (0.08) 0.255 −0.0 (0.06) −0.0 (0.05) 0.400 Lean mass arms (kg) 13 2.3 (0.67) 2.3 (0.41) 0.937 15 2.3 (0.44) 2.2 (0.41) 0.049 0.0 (0.36) −0.1 (0.24) 0.088 Lean mass legs (kg) 13 6.8 (1.7) 6.9 (1.45) 0.630 15 6.4 (1.08) 6.5 (1.04) 0.260 0.1 (0.64) 0.1 (0.34) 0.756 Lean mass trunk (kg) 13 23.0 (4.83) 22.6 (4.47) 0.212 15 21.5 (3.88) 21.7 (3.61) 0.198 −0.4 (1.21) 0.2 (0.67) 0.128 Total lean mass (kg) 13 45.2 (9.85) 44.7 (8.54) 0.545 15 42.3 (6.63) 42.5 (6.61) 0.458 −0.4 (2.52) 0.2 (1.02) 0.611 Fat mass arms (%) 13 2.6 (0.96) 2.6 (0.85) 0.808 15 2.4 (0.77) 2.3 (0.92) 0.291 −0.0 (0.56) −0.1 (0.41) 0.575 Fat mass legs (%) 13 5.8 (1.85) 5.9 (2.07) 0.165 15 5.4 (1.84) 5.5 (1.69) 0.506 0.2 (0.45) 0.1 (0.46) 0.549 Fat mass trunk (%) 13 17.1 (3.86) 16.7 (3.31) 0.448 15 14.9 (3.03) 15.7 (2.61) 0.061 −0.4 (1.86) 0.7 (1.31) 0.297 Total fat mass (%) 13 35.4 (8.05) 35.2 (7.53) 0.728 15 32.1 (6.84) 32.7 (6.64) 0.092 −0.2 (1.91) 0.6 (1.31) 0.357 Nutritional status MNA score 13 23.1 (3.8) 25.3 (2.2) 0.010 15 24.5 (2.1) 26.2 (1.6) 0.019 2.2 (2.6) 1.7 (2.5) 0.512 Normal nutritional status (N. %) 13 4(30.8) 9(69.3) 0.123 15 11(73.3) 14(93.4) 0.533 At risk of malnutrition (N. %) 13 8(61.6) 4(30.8) 15 4(26.7) 1(6.7) Malnourished (N. %) 13 1(7.7) 0 15 0 0 Biomarkers Creatinine (mg/dL) 10 1.1 (0.48) 1.1 (0.37) 0.664 15 0.9 (0.35) 0.9 (0.32) 0.595 0.0 (0.21) 0.0 (0.14) 0.438 Albumin (g/dL) 13 3.9 (0.39) 4.1 (0.31) 0.189 15 3.9 (0.31) 4.0 (0.26) 0.499 0.1 (0.22) 0.0 (0.15) 0.331 Prealbumin (mg/dL) 12 22.2 (6.63) 20.5 (4.48) 0.221 14 23.3 (4.31) 21.3 (4.17) 0.019 −1.6 (4.36) −1.9 (2.77) 0.916 SFT chair stand test 30sec: Senior Fitness Test chair stand test 30 sec; SFT arm curl test 30 sec: Senior Fitness Test arm curl test 30sec; SFT 6MWT (m): Senior Fitness Test 6-min Walking Test (m); SPPB total punctuation: Short Physical Performance Battery total punctuation; SPPB 5Squat: Short Physical Performance Battery 5Squat; BMI: body mass index; MNA score: Mini Nutritional Assessment score. Values are means and standard deviations. *P indicates statistical differences between Pre and Post values (paired Student’s t-test). ∆ placebo indicates the difference between Pre and Post values in the Placebo-group; ∆ Protein indicates the difference between Pre and Post values in the Protein-group. Pindicates statistical significance between ∆placebo and ∆Protein (ANOVA). Nutrients 2019,11, 2337 9 of 15 3.2. Effects of the Intervention on Secondary Outcomes We did not observe any significant difference on body composition measurements within groups at the end of the intervention, except for lean mass on arms within the protein-group (p<0.05, Table 3). There were no significant differences in any of the body composition variables between the two groups (Table 3). The MNA scoring improved significantly within both groups after the intervention program (p <0.05, Table 3). However, we did not observe any significant difference on changes in MNA score between groups (p<0.5, Table 3). Among serum markers of protein malnutrition, creatinine and albumin concentrations did not significantly change over time in either group (Table 3). Prealbumin concentrations significantly decreased in the Protein-group (p<0.05, Table 3). Nevertheless, there were no significant differences on changes in protein nutritional status serum biomarkers between groups (Table 3). 4. Discussion The current study aimed to examine the additional effect of a leucine-enriched protein supplementation on physical function, skeletal muscle mass and nutritional status after resistance training in a post-hospitalized elderly population. Results do not show further beneficial effects with protein and leucine-enriched supplementation after 12 weeks of resistance training (2 sessions/week) for any of the measured variables. These findings suggest that protein supplementation might not be determinant to see improvements in muscle mass and strength, and/or the time period of the intervention was not enough to see significant results. It is well-established that resistance training is an effective countermeasure to combat age-related skeletalmuscle mass andstrengthloss[ 6 , 29 ]. Itis proposedasa primaryinterventionfor sarcopenia[ 30 ], frailty [ 31 ], malnutrition [ 32 ] and other geriatric syndromes [ 7 ]. Our results are in line with these guidelines, according to physical function measurements as both groups show improvement after resistance training. Resistance training stimulates muscle protein synthesis [ 33 ]. To take advantage of this anabolic stimuli, we considered protein supplementation as a complementary strategy following resistance training. In line with studies supporting this strategy [ 15 , 34 ], the protein-group received 20 g of whey protein enriched with 3 g of leucine after each session twice per week. However, there were no further benefits on physical function for the protein-group in this study. This is in contrast with some [ 34 – 36 ], but not all [ 37 , 38 ] previous studies. A recent systematic review [ 18 ], concluded that protein/AA supplementation did not further improve muscle strength in older subjects following a resistance training program. Nevertheless, both groups showed significant improvements in physical function parameters, except for handgrip strength. This result was also seen in the study carried out by Leenders et al. [ 29 ], where they suggested that handgrip strength is not a clinically relevant and/or valid measure to evaluate changes in muscle function in response to a resistance training program in the elderly. We observed no changes in body composition after the intervention in either group. Again, we did not see further benefits with protein supplementation. One previous study with participants aged 82 years reported a limited muscle plasticity that further limited strength gains in response to a progressive resistance training program [ 39 ]. So, our results in a population with the same average age (82 years) underscore the limited capacity to hypertrophy as we age [ 40 ]. Furthermore, when looking for studies regarding muscle mass and strength gains along with protein supplementation, among many of them, the target adult population are younger than age 80 [ 34 , 36 , 41 – 43 ]. The same issue can be seen in recent systematic reviews and meta-analyses, where most of the included studies are based in younger populations [ 44 , 45 ]. However, this blunted anabolic response might be overcome, or at least minimized, if adequate interventions are designed [ 46 ]. It seems that the protein synthesis capacity of the muscle is preserved up to very old age in response to anabolic stimuli [33].