Increasing participation rates and improving performances of masters endurance athletes (14) warrant an improved understanding of potential differences in postexercise recovery between younger and older athletes (10,26). A better understanding of recovery after intense exercise, and the underlying mechanisms, is likely to inform more effective nutritional and recovery practices for masters athletes and, in turn, further improve training adaptation and competition performance. The limited available data suggest that masters endurance athletes recover at similar rates to younger athletes following fatiguing cycle exercise (3,11). However, it appears that older athletes take longer to recover when compared with younger athletes after running, which is associated with greater muscle damage (9,10).
After exercise-induced muscle damage, longer recovery durations required by masters compared with younger athletes may be due to age-related impairments in the repair/remodeling mechanisms in skeletal muscle. In older untrained adults, there is an age-related “anabolic resistance” after both resistance training (13) and protein feeding (19,21), with lower muscle protein synthesis (MPS) rates often observed in response to these stimuli compared with younger adults. Despite suggestions that regular physical activity might attenuate age-related decrements in MPS by reinstating anabolic sensitivity to aminoacidemia (6), this has yet to be confirmed. In the present investigation, we chose to study older well-trained masters triathletes as a model of highly active aging, because their habitual high levels of regular exercise should, in theory, offset age-related anabolic resistance.
The aim of this study was to compare the myofibrillar fractional synthetic rate (FSR) of well-trained masters and younger triathletes over a 72-h period of intense endurance training after a downhill run. We also, as an applied test of the capacity for recovery, compared potential age-related differences in the recovery of endurance cycling performance after the muscle-damaging run using multiple cycling time trials (TT) at 10, 24, and 48 h postrun. We hypothesized that masters triathletes would exhibit lower myofibrillar FSR in comparison with younger triathletes, and that masters triathletes would recover their performance at a slower rate than younger triathletes.
Six young (27 ± 2 yr) and five masters (53 ± 2 yr) well-trained triathletes participated in the present study. All reported training ≥10 h·wk−1 for at least 8 wk before the study. Table 1 outlines participant characteristics of each group. The study was approved by Central Queensland University’s Human Research Ethics Committee and conformed to all international standards: Helsinki declaration and the Canadian Tri-Council Policy on the participation of humans in research. All participants gave written informed consent before participation in the study.
Participants visited the Exercise Physiology Laboratory at Central Queensland University for preliminary testing (V˙O2max testing), and 48 h later, they completed a baseline 20-km cycling TT. At least 5 d later, participants completed the 3-d experimental trial. Baseline testing was conducted at the same time of day as morning sessions of the experimental trial (0400–0800 h). All exercise bouts were completed under standard laboratory conditions (22°C–24°C, 60% relative humidity).
Participants completed a preexercise screening, a training questionnaire, anthropometric measures, and a V˙O2max test. Skinfolds were obtained from eight sites using calibrated skinfold calipers (Harpenden; Baty International, West Sussex, UK), and body fat percentage was calculated (32). Subsequently, V˙O2max testing was completed on a motorized treadmill (TMX428; Trackmaster, Newton, KS) commencing at a speed of 8 km·h−1 and increased at a rate of 1 km·h−1·min−1 until volitional exhaustion. Expiratory gas was continuously analyzed via a metabolic cart calibrated to the manufacturer’s instructions (Trueone 2400; Parvomedics, Sandy, UT). V˙O2max was determined as the highest rate of oxygen consumption recorded over an averaged 15-s period. Participants were subsequently familiarized with all experimental protocols, including a truncated (60%) 20-km cycling TT under experimental conditions.
A schematic overview of the experimental trial can be found in Figure 1. On day 1, participants reported to the laboratory after an overnight fast. Baseline and resting skeletal muscle, and saliva were sampled. Blood was sampled for the analysis of creatine kinase (CK) activity, and participants consumed a 150-mL bolus of deuterium oxide (D2O). One hour later, and after a 6-min warm-up, participants completed a 30-min downhill run (−10%) on a motorized treadmill at a speed corresponding to 70% of V˙O2max during flat running (4). Participants returned to the laboratory 10 h postrun and provided a blood sample for CK analysis. They then completed a standardized 6-min warm-up on a cycle ergometer before completing a 20-km cycling TT. This afternoon testing format was repeated at 24 and 48 h after the downhill run. Saliva was collected upon arrival to the laboratory each morning, and skeletal muscle was sampled at 72 h after the initial biopsy. Both were used in the assessment of MPS.
Diet and exercise standardization
Participants completed a 3-d diet record before testing. Based on the data provided, individualized diets for the 24 h preceding the baseline TT and for the period spanning 24 h pretrial until the final muscle biopsy at 72 h were prescribed (Foodworks 7.0®; Xyris, Brisbane, Australia), and all food consumed was recorded. Prescribed carbohydrate intake was 6 g·kg−1·d−1 (7 g·kg−1·d−1 on experimental day 1), and protein intake was ~1.6 g·kg−1·d−1 (~1.7 g·kg−1·d−1 on experimental day 1) with each meal containing 0.3 g·kg−1 of protein. Water was consumed ad libitum throughout the study; however, this was not recorded. Immediately after each exercise bout, participants were provided with an individualized protein–carbohydrate beverage consisting of 20 g of protein (Whey Protein Isolate 894, Fonterra, Australia) and 1 g·kg−1 carbohydrate (1/1 of maltodextrin/glucose) to consume over the first hour of recovery. After all morning exercise bouts, a second carbohydrate-only beverage (1 g·kg−1) was provided to the participants; they were instructed to consume this beverage over the second hour of recovery. Participants were required to abstain from caffeine intake 12 h before each exercise bout, and to refrain from vigorous exercise 48 h before both the baseline testing and experimental trial, and all exercise 24 h before testing. Only exercise associated with the experimental trial was completed in the 3-d trial period.
Participants arrived at the laboratory after an overnight fast and rested supine for 15 min. Local anaesthetic (1% lidocaine) was administered to the lateral aspect of left vastus lateralis, and muscle was sampled using a 14-gauge percutaneous needle biopsy with cannula (Achieve®; CareFusion, Seven Hills, NSW, Australia). Approximately 80 mg of muscle was sampled, snap frozen in liquid nitrogen, and stored at −80°C until subsequent analysis. Final muscle (72 h) samples were obtained from the contralateral leg.
Immediately after baseline skeletal muscle and saliva sampling, each participant consumed a single 150-mL bolus of 70% D2O (Cambridge Isotope Laboratories, Tewksbury, MA). This bolus has been shown to label body water at ~0.2 atom percent excess (APE) by 24 h postingestion and has been shown to result in a linear incorporation of 2H into muscle-bound alanine over a period of ≤4 d (27). Saliva samples were obtained daily, at least 30 min after consumption of any food or drink, for measurement of 2H in body water. 2H body water enrichment was used as a surrogate for plasma alanine 2H labeling (2,27).
Skeletal muscle (~50 mg) was homogenized in ice-cold buffer, and the myofibrillar fraction separated as previously described (7). Analysis by gas chromatography pyrolysis isotope ratio mass spectrometry was conducted (Metabolic Solutions, Nashua, NH) as described elsewhere (2). The rate of myofibrillar FSR (%·d−1) was calculated by the standard precursor–product method (27). Briefly,
where EAla is the enrichment of muscle-bound alanine (APE) at each respective time point (1 = baseline and 2 = posttrial), EBW is the mean 2H enrichment of body water (APE) between time points (27), t is the tracer incorporation time in days, 3.7 is the mean number of 2H atoms incorporated into alanine (15,27), and multiplication by 100 converts the fraction to a percentage.
On each occasion (10, 24, and 48 h postrun), participants completed a 20-km cycling TT (Velotron Dynafit Pro; RaceMate, Seattle, WA). The 20-km TT has been shown to be highly reproducible (coefficient of variation = 0.7%) in trained cyclists (23). Feedback regarding distance covered at 25%, 50%, 75%, and 90% of completion was provided.
CK activity was measured in capillary blood with a clinical chemistry system (Reflotron Plus; Roche Diagnostics, Almere, The Netherlands) as per manufacturer’s instructions. This system has been reported to have high within-series and between-day precision with a coefficient of variation of 3.1% and ≤3.0%, respectively (12).
Data are presented as mean ± SD unless otherwise stated. Data were analyzed using SPSS (Version 22.0; SPSS, Armonk, NY). Normality was assessed by skewness and kurtosis z-scores. Independent t-tests were used to compare between-group differences for demographic data and myofibrillar FSR. TT performance and CK activity were examined over time using repeated-measures ANOVA. Within-group, between-time-point comparisons of TT performance were assessed by multiple paired t-tests with Bonferroni corrections. Alpha was set at 0.05. Cohen’s d effect sizes were also determined for all analyses and used to compare the magnitude of change in TT performance. Threshold values for small, moderate, and large effect sizes were 0.2, 0.5, and 0.8, respectively (25).
Figure 2 shows saliva 2H enrichment (APE) decayed in a manner that was well described by linear models over the 3-d trial.
Figure 3 shows (A) myofibrillar FSR (%·d−1) over the 3-d period and (B) muscle 2H enrichment (APE) at 72 h in masters and younger triathletes. Masters triathletes had significantly lower muscle 2H enrichment (P = 0.03) and myofibrillar FSR compared with younger triathletes (1.49% ± 0.12%·d−1 and 1.70% ± 0.09%·d−1, respectively; P = 0.009; d = 1.98).
Figure 4 shows the change in TT performance time (s) relative to baseline performance at 10, 24, and 48 h after the downhill run. Although there were no statistical differences (P > 0.05), there was a trend for masters triathletes to recover more slowly, indicated by a moderate between-group effect (d = 0.51) when comparing change in performance time between the baseline and the 10-h TT. Between-group effects for the change in performance time between the baseline and the 24-h TT, and the baseline and the 48-h TT, were small (d = 0.20) and trivial (d = 0.08), respectively, and likely due to high variability in TT performance among masters triathletes.
Figure 5 displays CK activity (U·L−1) for masters and younger triathletes at the baseline, and at 10, 24, 48, and 72 h postrun. CK activity was not different between groups, suggesting similar muscle damage over the 3 d. No group or interaction effect was observed (P > 0.05) for CK activity; however, a significant effect of time (P < 0.001) was observed.
This study is the first to compare integrated MPS rates and exercise performance of well-trained masters and younger endurance athletes over 3 d of consecutive training after a muscle-damaging bout of running. Our data show that masters triathletes had a lower myofibrillar FSR compared with younger triathletes when 20 g of dietary protein was provided postexercise, and protein was prescribed during the study period at doses suggested to maximize MPS among younger athletic cohorts (24). We also observed a trend, as suggested by a moderate between-group effect size, for masters triathletes to experience greater decrements in afternoon cycling performance after a morning muscle-damaging run compared with younger triathletes.
The use of D2O to 2H isotope label newly synthesized muscle proteins has several favorable benefits over traditional acute stable isotope infusions. For example, the ability to measure MPS over multiple exercise bouts and days (27), incorporating normal feeding practices (16), provide a “real world” measure. To date, no other studies have evaluated MPS rates in well-trained athletes, nor investigated the effect of age on MPS in well-trained athletes by use of this method. Therefore, comparison of our data with prior studies is difficult. However, studies using D2O for analysis of MPS in older untrained adults in response to aerobic exercise have reported comparable myofibrillar FSR with those in the present study (2). Furthermore, Wilkinson et al. (27) present similar 2H enrichments in body water 24 h postconsumption of D2O, with rates of decay similar to the present study over the initial 3-d period. Therefore, our data appear to be in line with previous studies using this approach to measure MPS.
In the present study, we observed that masters triathletes exhibited lower myofibrillar FSR over 3 d of consecutive training. Although resting MPS rates were not measured, these data have potential implications for the recovery of older athletes after exercise. Previous research has suggested that elevations in myofibrillar FSR may be a response to unaccustomed exercise (28), and may be an important response to repair damaged muscle (17,18) and to potentially facilitate a more rapid recovery after exercise (17). Despite the fact that age-related neuromuscular factors may contribute to slower recovery in older athletes (10), the lower myofibrillar FSR, and thus likely slower repair and remodeling of myofibrillar proteins observed in the present study, may contribute to a slower recovery in masters compared with younger athletes after eccentric muscle damage, as observed here and elsewhere (10). However, we cannot discount that a lower force production during the cycling TT by masters athletes may have lessened the mechanical stimulus for anabolic signaling and thus reduced the measured MPS response, given that this MPS rate is inclusive of these 3-d training. Nevertheless, attenuated cumulative elevations in MPS, as observed in masters athletes, could represent an attenuated adaptation to training (17,18).
The lower myofibrillar FSR observed among masters triathletes in the present study occurred despite postexercise and daily protein (per meal) consumed in accordance with current sport nutrition guidelines (24). Specifically, our triathletes ingested 20 g of high-quality, leucine-rich whey protein after each exercise bout in line with the current sport nutrition recommendations (1,24); furthermore, each main meal contained 0.3 g·kg−1 of protein, a dose suggested to maximize MPS among young healthy cohorts postexercise (20). Importantly, current sport nutrition recommendations do not differentiate between masters and younger athletes. Therefore, masters athletes are currently recommended to consume protein boluses, and report consumption, in line with those recommended for younger athletes (8); these protein doses were therefore implemented in the present study. Our findings suggest that aging, or the inability to produce youth-like force into later age, and not merely lower habitual physical activity, may attenuate MPS rates. However, there appears some potential for higher postexercise protein intakes to offset attenuated MPS response in masters athletes (19,21,30,31).
Collectively, studies that have examined the effect of postexercise protein feeding among young endurance athletes suggest that aminoacidemia significantly increases myofibrillar FSR, in a dose–response manner (5,22). However, in younger athletes, this dose–response relationship has a ceiling at doses of protein of approximately 20–25 g (22), with higher doses merely elevating amino acid oxidation (29).
In contrast, data from acute stable isotope infusions suggest that older untrained adults show a dose–response between protein and MPS up to protein doses of 40 g (30). For example, Robinson et al. (21) have shown that healthy middle-age men (59 ± 2 yr), of similar age to the masters triathletes in this study, showed a continued increase in myofibrillar FSR with consumption of 36 g of protein postexercise and at rest in comparison with boluses of 24 g or less, when consumed as beef. Similarly, Yang et al. (31) have shown that elderly men (71 ± 5 yr) elicit significantly greater MPS rates in response to 40 g of whey protein compared with 20 g of whey protein. Given the lower myofibrillar FSR observed in masters compared with younger triathletes in the present study, we suggest that habitually high levels of physical activity do not offset the need for higher age-appropriate doses of high-quality protein; this may be one strategy to counteract age-related attenuations in MPS and facilitate recovery and adaptation processes in masters athletes. Future research comparing MPS rates of masters athletes in response to habitual and higher protein intakes relative to baseline measures, in the presence and absence of exercise, should be investigated.
The present study also found that masters triathletes tended to have poorer same-day (10 h postrun) cycling performance (−3.0%, d = 0.46) after morning muscle-damaging exercise compared with younger triathletes (−1.4%, d = 0.29), as evidenced by a moderate between-group effect when comparing change in performance from the baseline. Few studies have examined recovery of exercise performance among masters compared with younger athletes after exercise-induced muscle damage. However, Sultana et al. (26) compared the recovery of physiological parameters among well-trained masters (52 ± 10 yr) and younger (28 ± 6 yr) triathletes after an Olympic distance triathlon. The researchers observed that masters triathletes had a significantly reduced run speed at ventilatory threshold 24 h postrace, whereas younger triathletes had recovered to baseline run speeds (26). It is reasonable to speculate that this would likely have translated into poorer endurance exercise performance, similar to the trend found in the present study. Importantly, the recovery durations investigated in the present study, and that by Sultana et al. (26), are similar to the normal training practices of competitive triathletes.
In summary, the present study has shown that well-trained masters triathletes have age-related attenuations in myofibrillar FSR compared with younger triathletes during a period of intense endurance training after muscle-damaging exercise. This finding was accompanied by a trend for masters triathletes to recover cycling performance at slower rates compared with younger triathletes. Future research examining the effects of higher protein intakes postexercise on MPS and recovery of exercise performance after muscle-damaging exercise among masters athletes are warranted.
The authors would like to thank Tracy Rerecich for her technical expertise and assistance.
This manuscript was funded by a postgraduate scholarship from the CQUniversity HEALTH CRN (www.cqu.edu.au/crn) and the Australian Government’s Collaborative Research Networks Program awarded to T. M. Doering.
The authors declare no conflict of interest. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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