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Protein-containing nutrient supplementation following strength training enhances the effect on muscle mass, strength, and bone formation in postmenopausal women

¹Institute of Sports Medicine, Bispebjerg Hospital, Copenhagen, Denmark; ²Saga Nutraceuticals Research Institute, Otsuka Pharmaceutical, Saga, Japan; ³Research Unit, Department of Anaesthesiology, Ribe County Hospital Esbjerg, Esbjerg; and ⁴Research Centre of Ageing and Osteoporosis, Department of Geriatrics, Glostrup University Hospital, Glostrup, Denmark

Submitted 3 September 2007 ; accepted in final form 29 April 2008

	ABSTRACT

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We evaluated the response of various muscle and bone adaptation parameters with 24 wk of strength training in healthy, early postmenopausal women when a nutrient supplement (protein, carbohydrate, calcium, and vitamin D) or a placebo supplement (a minimum of energy) was ingested immediately following each training session. At inclusion, each woman was randomly and double-blindedly assigned to a nutrient group or a placebo (control) group. Muscle hypertrophy was evaluated from biopsies, MRI, and dual-energy X-ray absorptiometry (DEXA) scans, and muscle strength was determined in a dynamometer. Bone mineral density (BMD) was measured using DEXA scans, and bone turnover was determined from serum osteocalcin and collagen type I cross-linked carboxyl terminal peptide. The nutrient group improved concentric and isokinetic (60°/s) muscle strength from 6 to 24 wk by 9 ± 3% (P < 0.01), whereas controls showed no change (1 ± 2%, P > 0.05). Only the nutrient group improved lean body mass (P < 0.05) over the 24 wk. BMD responded similarly at the lumbar spine but changed differently in the two groups at the femoral neck (P < 0.05) [control: 0.943 ± 0.028 to 0.930 ± 0.024 g/mm³ (–1.0 ± 1.4%); nutrient group: 0.953 ± 0.051 to 0.978 ± 0.043 g/mm³ (3.8 ± 3.4%)] when adjusted for age, body mass index, and BMD at inclusion. Bone formation displayed an interaction (P < 0.05), mainly caused by increased osteocalcin at 24 wk in the nutrient group. In conclusion, we report that nutrient supplementation results in superior improvements in muscle mass, muscle strength, femoral neck BMD, and bone formation during 24 wk of strength training. The observed differences following such a short intervention emphasize the significance of postexercise nutrient supply on musculoskeletal maintenance.

exercise; nutrition; muscle hypertrophy

We (26) reported earlier that ingestion of a nutrient supplement immediately after a session of resistance exercise improves thigh net protein balance compared that following a placebo supplement. The possibility of additive bone adaptation with a similarly timed protein supplementation has not previously been investigated. Therefore, based on our earlier finding (26) and the uncertainties of how bone metabolism responds acutely to nutrient availability, we hypothesized that early postmenopausal women, daily consuming a weight-maintaining diet containing protein (36) and micronutrients, would demonstrate superior improvements in muscle mass, strength, and bone health when a small amount of nutrients was ingested immediately after single strength training sessions, compared with a control situation where a placebo supplement was given.

	METHODS

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Subjects.   More than 60 women responded to newspaper advertisements for the study, and 44 of these were invited for a clinical examination and individual information concerning the study in accordance with the Declaration of Helsinki II before written consent was given. During the clinical examination, some women were found not to meet the inclusion criteria, and some withdrew their commitment after the detailed information had been given. Thirty-eight women were included for double-blinded randomization into either the nutrient or placebo (control) group. During the intervention period, 9 of the 38 subjects dropped out for the following reasons: one developed depression, one developed knee pain, and seven realized with time that their daily routines did not permit adherence to the protocol. Hence, with 13 subjects in the nutrient group and 16 subjects in the control group, 29 healthy, early postmenopausal women completed 24 wk of resistance exercise training. All the women were on a weight-maintaining diet and, for the duration of the study, refrained from taking any medication and maintained their levels of physical activity.

Study design.   The study protocol was approved by the local ethics committee [reference number (KF) 01-177/01]. The difference in intervention between the nutrient and control groups was the content of the supplementation administered immediately after each training session. The nutrient group was supplied with 730 kJ, composed of 10 g of protein (whey protein), 31 g of carbohydrate, 1 g of fat, 5.0 µg of vitamin D, and 250 mg of calcium, whereas the placebo (control) group received 102 kJ as 6 g of carbohydrate and 12 mg of calcium. Both supplementation types were blinded with respect to taste (added artificial sweetener and taste additives). No food intake was allowed from 2 h before the start of training to 2 h after completion of the session. The supplementation was ingested immediately after each training session. Free intake of water was permitted.

Resistance exercise protocol.   After a brief warm-up on a Monarck cycle ergometer, the resistance exercise protocol was completed. The resistance exercise protocol consisted of two types of supine leg-press exercises: one with high-foot (H-F) position, similar to a squat movement, and another with low-foot (L-F) position. Repetitions (reps) and intensities were 3 x 15 reps at 20 repetition maximum (RM) for H-F and L-F exercises during period I (weeks 1 and 2), 3 x 10 reps at 10 RM for H-F exercise and 4 x 10 reps at 10 RM for L-F exercise during period II (weeks 3–12), and 5 x 8 reps at 8 RM for both exercises during period III (weeks 13–24). Furthermore, a knee extension exercise was conducted with the following progression: period I, 3 x 15 reps at 20 RM; period II, 4 x 10 reps at 10 RM; and period III, 3 x 10 reps at 10 RM. Hereafter, 10 reps at periods I and II and 20 reps at period III of sit ups and back extension exercises were conducted. Finally, each exercise session, throughout all three periods, ended with 1 x 15 reps at 20 RM o latissimus muscle pull-down exercise to strengthen the back. Subjects trained three times per week during periods I and II and twice a week during period III.

Food recording.   The seven women in each group who were consuming any supplementation at inclusion (Table 1) were requested to stop taking this for 1 mo before the start of training, and all participants were asked to refrain from taking any kind of supplementation during the intervention. Weighed food recording was carried out three times (at 0, 12, and 24 wk) for four nonconsecutive days, including a weekend day, which enabled us to monitor the subject's eating behavior. Each food recording was analyzed by the same person using the Ankerhus Kostprogram WinFood, version 2.0. Because of the double-blinding of supplementation type, the content of supplementation was not included in the original analysis of food intake. However, by adding one-seventh of three supplementations (control or nutrient) per week to the mean of three recorded days at the midtraining recording (after 12 wk) and one-seventh of two supplementations (control or nutrient) per week to the mean of three recorded days at the postintervention recording (after 24 wk of intervention) to the two groups, respectively, we calculated the total daily intake, including the content of the supplementation.

Muscle biopsy.   After the MRI scans and the strength tests were conducted but before the intervention started (and after 12 wk of training), we obtained a muscle biopsy from the vastus lateralis muscle at the location corresponding to the mid-CSA from the MRI scans. With subjects under local anesthesia, a 50- to 80-mg muscle specimen was obtained using a Bergstrom needle (8) with suction applied (17). The muscle piece was mounted in Tissue Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands), frozen in precooled isopentane, and kept at –80°C until analysis. The embedded muscle specimens were blinded, and transverse sections (10 µm) were cut at –21°C in a cryostat (Cryo-Star HM 560 M; Microm International, Walldorf, Germany). Mounted on glass slides, serially cut sections were histochemically stained for ATPase activity after preincubation at pH 4.37, 4.60, and 10.3 for differentiation and visualization of fiber types (14) using Tema software version 1.04.

Strength determination.   Muscle strength was measured in a knee extensor model similar to the strength training exercises in a KinCom dynamometer (model 500-11 Kinetic Communicator; Chattecx, Chattanooga, TN). After a separate day of familiarization to the strength-testing protocol and equipment, the pretraining measurement was conducted. Hereafter, strength recordings were obtained after 6, 12, and 24 wk of training. At each test round, the subjects initially warmed up for 5 min on a Monarck cycle ergometer, after which they were seated in a rigid chair and strapped cross the thigh, hips, and chest. Just before the maximal tests, a few submaximal attempts were conducted for familiarization, after which three or, if necessary, more attempts of maximal contraction were completed with more than 30 s of relaxation in between. Visual feedback from a monitor and strong verbal encouragement were given during each test and each contraction. Test velocity was 60°/s, and the strength was measured concentrically in the range of motion from 90° flexion to 10° (with 0° as full extension).

Dual-energy X-ray absorptiometry canning.   Before and after training intervention, BMD and body mass were evaluated in all subjects with dual-energy X-ray absorptiometry (DEXA) scanning (Lunar DPX-IQ software version 4.6 c; Lunar, Madison, WI). Subjects were instructed not to conduct any strenuous work or exercise for 2 days before the scans and arrived for scanning in a postabsorptive and euhydrated state. Total body, lumbar spine, and hip scans were performed in the same scanning session. The subjects were placed in the supine position, and a whole body scan was performed using the medium scan mode and extended research analysis. Body composition was evaluated from the whole body scan. Lumbar spine and hip (single side) scans were performed using the standard protocols from Lunar. BMD was measured in the total body, at the lumbar spine (L2–L4), and total femoral region. All the scan sites were conducted as single determinations. In all cases, all vertebrae (L2–L4) were readable without artifacts from osteoarthritis or fractures. The DEXA scanner was calibrated on the morning of each day of the study using the recommended daily calibration procedure (Lunar "Quality Assurance").

Collagen type I cross-linked carboxyl terminal peptide and osteocalcin.   Two serum markers of bone remodeling status (18, 31), osteocalcin and collagen type I cross-linked carboxyl terminal peptide (CTx), were measured in the fasted state at 0, 12, and 24 wk. Osteocalcin is the most abundant noncollagenous protein in bone. During synthesis of bone, a small fraction of newly synthesized osteocalcin is released into the circulation. Thus we measured serum osteocalcin concentrations with a One Step ELISA (Nordic Bioscience Diagnostics, Herlev, Denmark), with inter- and intra-assay coefficients of variation of 4.1 and 2.0%, respectively, as an indirect measure of bone formation. CTx, originating from collagen I degradation, was measured as an indirect measure of bone resorption with a CTx One Step ELISA (Nordic Bioscience Diagnostics), with inter- and intra-assay coefficients of variation of 5.4 and 5.0%, respectively.

Vitamin D and parathyroid hormone.   Vitamin D and parathyroid hormone (PTH) status were determined from fasting blood samples at 0, 12, and 24 wk. Vitamin D is a key regulator of bone turnover. Vitamin D status consists of the 25-hydroxy precursor of the more bioactive component 1,25-dihydroxy. Concentrations of vitamin D, 25-hydroxy, and 1,25-dihydroxy were quantified from serum samples by using RIA kits (Gamma-B 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D RIA kits; Immuno Diagnostic Systems, Biotech-IgG, Copenhagen, Denmark), with inter- and intra-assay coefficients of variation of 8.1 and 5.0%, respectively, for the 25-hydroxy vitamin D kit and 9.5 and 5.0%, respectively, for the 1,25-dihydroxy vitamin D kit. By regulating the conversion of 25-hydroxy to 1,25-dihydroxy vitamin D, PTH is an important player in the bone metabolism. PTH in plasma samples was measured using a RIA kit (DPC Scandinavia, Køge, Denmark), with inter- and intra-assay coefficients of variation of 5.8 and 4.2%, respectively. Regarding the 25-hydroxy vitamin D concentrations, which are known to be seasonal dependent, the 0- and 12-wk blood samples were obtained in November or December and in March and April, respectively, when Danish sunlight exposure is very limited. The 24-wk blood samples were taken from the end of May till mid-July with much warmer weather and much more sunlight exposure.

Statistics.   The approximate sample size was determined using the Altman nomogram: power at 80%, significance level at 5%, and the standardized difference, estimated to be 1, calculated as the ratio of standard deviation of one outcome parameter (muscle hypertrophy determined by MRI scans) over the clinical relevant difference. Sample size was found to be around 30 subjects, thus 15 subjects in each group. The results are means ± SE. Parametric tests were conducted, since the data sets were assumed to be subpopulations of Gaussian distributed variables. A two-way ANOVA with repeated measures was used to compare the two groups when three or more data points were collected over time. When the outcome was significant, the Fisher's least significant difference (LSD) post hoc test was applied for paired comparisons. We applied t-tests when only two data points were measured. A between-groups analysis of covariance was conducted to compare the effect of the nutritional intervention in increasing muscle strength and BMD. The independent variable was the type of intervention (nutritional vs. placebo), and the dependent variable consisted of improvements in the velocity-specific (60°/s) isokinetic training strength and changes in BMD in total body, femoral neck, and the lumbar spine. SigmaStat version 3.5 software (Systat Software, San Jose, CA) and SPSS version 11.5 software (SPSS, Chicago, IL) were used. Significance level was chosen as P < 0.05.

	RESULTS

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Baseline characteristics of participants.   Both groups were similar at baseline with regard to the measured parameters, with the exception of body mass index (BMI), where the controls had a slightly higher BMI than the nutrient group (27 ± 1 and 24 ± 1 kg/m, respectively; Table 1).

Training compliance.   The two groups were statistically comparable with respect to training intensity. During periods I and II, mean training frequency in the control and nutrient groups was 2.61 ± 0.09 and 2.75 ± 0.05 times per week, respectively (P > 0.05), and intensity was 1.82 ± 0.03 and 1.88 ± 0.05 times per week, respectively (P > 0.05), during period III.

Nutrient intake.   Daily energy and protein intake per kilogram of fat-free mass were equal between groups at inclusion and at 12 and 24 wk regardless of whether the comparison was made with or without the content of the supplementation. Thus it cannot be the nutrient content per se that the supplement adds to the daily intake that is responsible for the improved exercise response in the nutrient group compared with the control group.

Calcium intake was not different between groups when the content of the supplement was excluded (data not shown), but a nutrient effect was apparent when the supplement content was included (see Table 2). The group means of daily calcium intake in the control group at 0, 12, and 24 wk and in the nutrient group at 0 wk were not significantly different from 800 mg, which is the general recommendation of daily calcium intake for this age group. However, the nutrient group had a calcium intake significantly higher than 800 mg at 12 and 24 wk, irrespective of the addition of calcium from the supplementation.

Whole muscle hypertrophy.   Both groups increased the quadriceps muscle CSA significantly at proximal, mid, and distal locations during the first 12 wk of intervention (see Table 3). However, there were no significant differences between the relative group changes at any of the locations. For the control and nutrient groups, respectively, the increases in CSA at three locations were as follows: proximal, 4.3 ± 1.0 vs. 6.0 ± 1.3%; mid, 5.0 ± 0.9 vs. 5.6 ± 0.7%; and distal: 5.4 ± 1.3 vs. 6.1 ± 0.9% (vastus medialis, 4.9 ± 1.1 vs. 5.2 ± 0.7%; vastus lateralis, 6.3 ± 3.1 vs. 9.6 ± 4.2%).

Strength.   The velocity-specific (60°/s) isokinetic training strength improvements revealed an effect of training (P = 0.003) and nutrition (P = 0.08) by a two-way ANOVA with repeated measures (Fig. 1). The post hoc tests revealed significant improvements in the nutrient group, as shown in Fig. 1, between 0 and 12 wk (P = 0.043), 0 and 24 wk (P < 0.001), 6 and 24 wk (P = 0.006), and 12 and 24 wk (P = 0.046), whereas it was only in the control group that a trend toward improved strength was observed during the initial 6 wk (P = 0.058). The relative strength improvements from 0 to 24 wk in the nutrient group at 14 ± 4% tended (P = 0.098) to be different from the change in the control group at 8 ± 4%. It is well recognized however, that during the very first week of resistance training in novice strength trainers, muscular strength increases markedly due to improvements in neural firing and motor control. Therefore, we chose to evaluate the strength improvements from 6 wk onward as a more lean measure of hypertrophy-induced muscle function. From 6 to 24 wk, we found that a significant difference was apparent between groups: nutrient group, 9 ± 3% vs. control, 1 ± 2% (P < 0.05). After adjusting for covariates [age at inclusion, BMI at inclusion, and the preintervention isokinetic training velocity (60°/s)], we still found a significant difference in the response to training from 6–24 wk in the two intervention groups, although we also found a significant influence of the covariates on the change in strength.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Quadriceps muscle strength. Isokinetic training at a velocity of 60°/s in a range of motion from 90° to 10° knee angle. Data are means ± SE. Training effect: P = 0.003 and interaction: P = 0.12 in a 2-way ANOVA with repeated measures. Markers below lines are for the placebo group (CON), and those above the line are for the nutrient group (NUT). ()P < 0.10; P < 0.05; P < 0.001 compared with 0 wk within the same group. ##P < 0.01 compared with 6 wk. $P < 0.05 compared with 12 wk.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Mass of body compartments measured using dual-energy X-ray absorptiometry (DEXA) scans before (0 wk) and after (24 wk) the intervention. Data are means ± SE. #P < 0.05 compared with 0 wk. FAT, body fat mass; LEAN, lean body mass.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Plasma osteocalcin and collagen type I cross-linked carboxyl terminal peptide (CTx) concentrations at 0, 12, and 24 wk of intervention. Data are means ± SE. A: osteocalcin. A significant (P < 0.05) interaction (2-way ANOVA with repeated measures) was apparent with NUT 24 wk being higher than NUT 0 wk [()P = 0.056] and CON 24 wk being numerically lower than 0 wk (P = 0.26). #P < 0.05; ##P < 0.01 compared with corresponding CON value. B: CTx. No significant changes, group differences, or changes over time were apparent.

	DISCUSSION

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Skeletal muscle adaptation.   After the initial 12-wk period of training plus supplementation intervention, no measurable effect of nutrient timing could be detected with regard to muscle hypertrophy when this was determined as whole muscle CSA. The relative hypertrophy of 4–6% following 12 wk of progressive, resistance exercise training intervention, which apparently occurred irrespective of nutrient supplementation, was less than what has been reported generally in young (1, 22) and in some older individuals (21, 48) but was similar to what has been found in other studies in the elderly (19, 20, 23, 42).

Our initial loading intensity during period I (which was the first 2 wk of the first 12-wk period; see Resistance exercise protocol in METHODS) may have been too light a stimulus for substantial muscle accretion. Therefore, 10 wk of heavy weight training were conducted with the potential to induce muscle hypertrophy. Despite the fact that 3 wk of heavy resistance training were recently shown to be sufficient to induce significant muscle hypertrophy in young people (46), we exercised a group that seemed to demonstrate a diminished responsiveness and maybe with a suboptimal exercise program. That we could not detect single muscle fiber hypertrophy does not necessarily rule out that it was present, since the variation of single-fiber analysis from small biopsies is relative high (12, 34), which makes small relative differences hard to detect.

Acknowledging the inherent variation in single-fiber analysis, we believe that a small but significant hypertrophy may have taken place over the first 12 wk of training intervention as determined by MRI scans. In young individuals as well as in older men, the nutrient effect on muscle hypertrophy, at both whole muscle and single fiber level, has been found to be detectable after 12 wk of high-intensity resistance training (16, 25). Our earlier study suggested that early postmenopausal women were less responsive to nutrients than young people but that they did gain a positive effect of nutrient supplementation on muscle mass accretion compared with placebo intake (26). Therefore, the combination of a relatively low level of hypertrophy found in the present study due to a short and suboptimal training period and a diminished nutrient responsiveness in the subject group may explain the lack of measurable nutrient effect after the initial 12-wk intervention. Unfortunately, because of technical and logistical reasons, we did not determine the quadriceps muscle area again by MRI or the single-fiber area after 24 wk. Hence, we cannot directly confirm that the long-term effect of nutritional supplementation in fact had a major beneficial effect on quadriceps muscle hypertrophy measured by MRI. However, we conducted the DEXA scan after 24 wk. Despite the lesser sensitivity on lean body mass (LBM) measurements from the DEXA scans compared with single muscle cross sections from the MRI scans, we observed a significant gain in LBM following 24 wk in the nutrient group but not in the control group (Fig. 2). In addition, the fact that the LBM from the DEXA scanning is on a whole body level and the exercise intervention mainly stimulated the legs implies a robust effect of the nutrient supplementation on muscle mass.

In support of a temporal additive effect of nutrient supplementation on muscle gain, we detected a more marked improvement in the training-related functional muscle strength in the nutrient group compared with the control group (Fig. 1). Changes in strength measured concentrically at a specific training velocity have been shown to reflect changes in muscle mass when the training period exceeds the initial weeks of training. Thus, in the initial period from pretraining to 6 wk, the neural adaptation component (20, 23) is presumably dominating the strength changes at 7% in the present study. Even in studies where hypertrophy was measurable in such early periods, the strength improvements still far exceeded mass gains (46, 50). Thus, to distinguish the hypertrophy-induced strength improvements from each other when comparing the two intervention groups, we looked at the changes in strength from 6 wk to 12 and 24 wk. Our data demonstrated a significantly larger improvement in strength in the nutrient group compared with the control group at 24 wk. Although attenuated compared with younger individuals, our findings on muscle mass and strength support our earlier conclusion (26) that skeletal muscle of early postmenopausal women has the potential to produce a stronger anabolic response to resistance exercise training when nutrients are supplied immediately after single training sessions, compared with a nonsupplemented training group.

Bone adaptation.   In both groups we found a significant improvement in BMD at the lumbar spine (trabecular bone) following 24 wk of intervention, which is in accordance with comparable training interventions (33, 38). In contrast, no adaptations were found at a whole body level or at the femoral neck when the raw data were compared over time (see Table 4). Although some disagreement exists, a recent meta analysis revealed that exercise alone does not enhance BMD at the femoral neck (29). However, our data revealed that when the adaptation at the femoral neck was adjusted to inclusion variables for age, BMI, and BMD, the control and nutrient groups responded differently (P < 0.05). This finding suggests that the responsiveness of the more slowly turning over cortical bone present at the femoral neck is sensitive to whether nutrients are available in the acute period after stimuli. To our knowledge, these data are the first to describe this dependency over time, despite the fact that the 24-wk intervention period in the present study may be just at the detection level to reveal the differences. The acute responsiveness, however, has been shown by Babraj et al. (4), who, using the stable isotope incorporation technique, demonstrated improved bone collagen synthesis rate following a bolus of protein and carbohydrate at rest, which is in accordance with the fact that protein has been ascribed to be important for bone health (13). Serum levels of osteocalcin were earlier reported to decrease during periods of training without any exercise-timed nutrient supplementation (32), which is somehow comparable to our control group (no change, P = 0.26). However, making nutrients available causes the fasting osteocalcin levels at rest to increase (P = 0.056 for the nutrient group), which was similarly demonstrated in a 6-mo training and protein supplementation intervention in young men and women, where the serum marker for bone formation (bone alkaline phosphatase, BAP) improved over time (5). Thus our findings of a significant interaction in osteocalcin level with time, combined with the unchanged level of the bone (collagen) resorption marker (CTx), suggest a positive effect of exercise-timed nutrient availability in order for cortical bone to respond positively to loading.

Nutrients and hormones.   No group differences were found between any of the hormones (see Table 5). The significant time effect on 25-hydroxy vitamin D is most likely due to the seasonal variation in Denmark (39), since subjects were included during the winter and ended during the summer. However, this change was similar in both groups and will not be discussed further. PTH and 1,25-dihydroxy vitamin D were found to be unchanged over time. Since subjects also recorded an adequate calcium intake, which has been shown to be crucial for obtaining the beneficial effect from HRT (47) and physical activity (37) on bone turnover and formation, we conclude that the hormonal system might not have contributed to any of our observed group differences.

With regard to nutrition, we assessed the subjects’ total energy intake to be sufficient for their individual sizes, and we assessed them to be weight stable (Fig. 2). Regarding energy and protein intake, the content of the supplement added a negligible amount to the subjects’ general intake, which is seen in the similarity of the statistical outcomes independent of the inclusion of the supplement in the analyses. Finally, the consumed amount of energy and protein in both groups met the amounts sufficient to allow a positive adaptation to training (15). The calcium intake, however, influenced the mean daily intake in the group comparisons. The relative differences were relatively small, however, compared with what is generally used as additional amounts (1,000 mg/day) showing minimal effects on bone health (43, 44). Bearing in mind that the control group ingested a calcium amount in accordance with the general recommendations, we do not ascribe the small extra daily amount of calcium in the nutrient group to be responsible for any of the changes seen in this group. The average daily intake of vitamin D was similar in the control and nutrient groups at inclusion. In the nutrient group only, vitamin D intake was significantly elevated at the 12-wk time point when the supplement was included in the analysis and at 24 wk irrespective of inclusion of the supplement. However, the different intakes did not have any impact on the systemic availability of either 25-hydroxy vitamin D or PTH, and if anything, the control group had a slightly elevated 1,25-dihydroxy vitamin D level (see Table 5). Therefore, with the uncertainties in the exactness of vitamin and mineral intakes based on 4 days of food recording, the fact that no effects on the available circulating levels were found (10), plus the supplied amounts recommended for significant beneficial effects on bone health as well as muscle function far exceed the group differences seen in our study [15–20 µg/day (9, 11, 40)], leads us to the conclusion that we do not ascribe the small group differences of daily intake of nutrients or vitamins to be responsible for the group differences found in the present study. Therefore, we believe that the observed group differences can be ascribed to the fact that the nutrient group ingested the supplement immediately after training and that the control group did not.

Conclusion.   Without being able to distinguish the specific effects of the different substrates in the supplementation, we can conclude that additional energy, protein, calcium, and vitamin D given in the immediate postexercise period exerts a small but significant additive effect on muscle mass and strength gains as well as a positive effect on bone mineral density at the femoral neck after a period of physical training. We were able to detect these changes over the relatively short intervention period of 6 mo. We suggest that these effects would be enhanced even further if applied over several years and would presumably result in a perceptible difference in health and function. Therefore, we recommend that resistance training individuals develop the habit of ingesting a supplement containing protein, vitamin D, and calcium immediately after each training session.

	GRANTS

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This work was supported by Saga Nutraceuticals Research Institute (Otsuka Pharmaceutical, Saga, Japan).

	ACKNOWLEDGMENTS

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We thank physiotherapist Lone Larsen for help with training the women, Prof. Per Aagaard for inspiring discussions of the strength data, and Dr. Lene Rørdam, Department of Clinical Physiology, for help with the DEXA scans.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Holm, Institute of Sports Medicine, Copenhagen, Bld. 8 1st, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark (e-mail: l.holm.isotope{at}gmail.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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