Impact of Long-term Endurance Performance on Muscles Stiffness in Marathon Runners Over 50 Years Old

The aim of this research is to evaluate marathon performance and asses the inuence of this long-distance running endurance exercise on the changes of muscle stiffness in recreational runners aged 50 + years. Thirty-one male long-distance runners aged 50–73 years participated in the experiment. The muscle stiffness of quadriceps and calves was measured in two independent sessions: the day before the marathon and 30 min after the completed marathon run using a Myoton device. The 42.195-km run was completed in 4.30,05 h ± 35.12 min, which indicates an intensity of 79.3% ± 7.1% of HRmax. The long-term, low-intensity running exercise (marathon) in older recreational runners, along with the low level of HRmax and VO2max showed no statistically signicant changes in muscle stiffness (quadriceps and calves). There was reduced muscle stiffness, but only in the triceps of the calf in the dominant (left) leg. Moreover, in order to optimally evaluate the marathon and adequately prepare for the performance training programme, we need to consider the direct and indirect analyses of the running economy, running technique, and HRmax and VO2max and DOMS variables. These variables signicantly affect the marathon exercise.


Introduction
Preparing long-distance runners, especially recreational runners and those over 50, to participate in marathons 1 requires a rational strategy of training 2 . This mainly applies to developing the runner' motor abilities, technical skills, and probably the two most important actions: tactical skills and adequate dietary supplementation during the marathon itself [3][4][5] . Modern long-distance training has to allow runners to sustain speci c loads of long duration. Therefore, continuous running, and increased fatigue may cause a runner to experience physiological changes that either enhance or diminish their performance or even make it impossible to continue the run 6-8 . Adequate planning of training for marathons involves selecting appropriate training methods, maintaining a rational relationship between training loads, competition loads 9 , and effective recovery, and applying appropriate pre-and postworkout supplementation 10 .
The majority of long-distance training programmes for a marathon are based on regular long, easy runs of between 20 and 40 km 11,12 . The main purpose of such training is to develop and/or maintain maximum aerobic power, which is the main requirement in order to successfully complete a marathon, regardless of the level of performance or age of the competitors 5,13 . In addition, according to Saunders et al 14 . long-distance runs are intended to enhance running economy (RE). This teaches the athlete to run at a pace 15 as e ciently as possible and translates into actual running pace during the competition 16, 17 .
Several researchers 18-20 claim that running economy (RE) is an aerobic demand for the maintenance of running and is referred to as the steady-state oxygen uptake (VO2) related to that speed 11 . It is known from practice that after such a long run the athletes experience a considerable amount of muscle damage and soreness 18 , which may adversely affect their muscle overload in the next training session 21 .
Long-distance running competitions are associated with high mechanical stress due to damage to various muscle bers, metabolic disorders, and muscle fatigue 22 . These elements have been attributed to stress that leads to muscle swelling and delayed muscle soreness (DOMS). It has been shown that disruption of muscle bers during DOMS, exacerbated by exercise, changes the mechanical properties of the muscles which causes muscle stiffness 23 . Increasing muscle stiffness impairs muscle function and, as a consequence, reduces the body's ability to continue exercising. From a physiological point of view, the level of muscle stiffness is strongly dependent on the size and architecture of the muscles [24][25][26] and their speci c structural functionality 27 . The physiological cross-sectional area (PCSA) was identi ed as one of the most important features determining muscle stiffness. Other determinants of stiffness are the type of muscle bres and the percentage of fast-twitch and slow-twitch bres, as the number and composition of bres, which determine the onset of fatigue and, indirectly, post-training stiffness 28 . Multiple training variables can affect muscle stiffness, including the type of muscle work performed, the functionality of the muscle used ( exors vs extensors), and the amount of effort taken until recovery.
There are no data on the level of muscle stiffness after prolonged exercise, especially running. In turn, it is known that in short-term, dynamic exercise, greater muscle damage causes large eccentric contractions, and that large eccentric muscle contractions during PT cause greater muscle damage than concentric ones 29,30 . This causes more delayed-onset soreness in muscles 31,32 .
Whilst the relationship between physiological or anthropometric variables and marathon nish-time have been widely investigated, no study to date has evaluated muscle stiffness's relative contribution to marathon performance. Therefore, despite the dozen marathon investigations, there is a lack of clarity as to the precise determinants of muscle stiffness on marathon performance. Therefore the recipe for success -completion of the marathon -remains somewhat elusive. Assessing muscle stiffness, when combined with other performance indicators previously analysed would bene t runners and coaches looking to improve their marathon performance. Therefore, the aim of this study was to evaluate marathon performance and asses the in uence of this long-term running endurance exercise on the changes of muscle stiffness in middle-aged marathon runners. We hypothesise that muscle stiffness will increase with the time that the marathon lasts, no matter what level of training the runner has at the moment.

Study design
The main objective of this study was to evaluate marathon performance and assess its in uence on the changes of muscle stiffness in middle-aged marathon runners. The muscle stiffness of quadriceps was measured in two independent sessions: the day before the marathon and 30 min after the completed marathon. Myoton measurements of each muscle group (12 points) were taken separately for the left and right legs.

Participants
Thirty-one male long distance runners aged 50-73 years participated in the experiment. Runners estimated their training experience as 10.61 ± 8.81 years on average. The average result of the marathon run for the study group was 4.30,05 h ± 35.12 min. All participants were free from any acute illness or chronic disease and did not take regular medication. The main division criterion was that the runners were over 50 years of age, and had participated in at least two marathons in the previous three years. An additional criterion was that all participants were actively training for long-distance running for at least one year. Before signing informed consent forms, the participants were informed about the aim of the experiment and the risk of injury. The study protocol was approved by the local Institutional Ethics Committee (Senacka Komisja ds. Badań Naukowych przy Akademii Wychowania Fizycznego we Wrocławiu). The research was conducted in accordance with the Declaration of Helsinki.

Marathon performance
The 37th PKO Wrocław Marathon (Wrocław, Poland, 19 September 2019) was organised by the City of Wrocław, Poland. Since the beginning of the run, The PKO Wrocław Marathon has been organised by the city of Wroclaw and is considered to be one of the largest running events in Poland. The PKO Wroclaw Marathon takes place annually in the beginning of September. The data on the running time was obtained from the electronic database of the marathon's organisers. It included the number of runners who started and nished the run, the individual identi cation number of the run, and the place and time of the run for each participant of the marathon. The individual runtime registered in the event was automatically measured using a radio frequency identi cation chip system. Intermediate times every 5 km were measured for the experimental group to accurately analyse the variability of their running pace. In addition, the heart rate (HR) was recorded using a monitor (Polar RS300X GPS; Finland) to examine each participant during the marathon run.

Applied equipment
The Myoton PRO (Myoton AS, Estonia and Myoton Ltd., London) is a wireless hand-held device that is placed perpendicular to the skin over the muscle being measured. This device was applied under constant preload (0.18 N) to pre-compress subcutaneous tissues and exert a brief (15 ms) mechanical tap at a predetermined force (0.4 N), followed by a quick release, thereby causing dampened oscillations that are recorded by the testing probe (http://www.myoton.com/en/Technology/Technical-speci cation). The non-neural tone or tension was calculated from the signal spectrum Fmax (fast Fourier transform [FFT]) and had the frequency (Hz) of the dampened oscillations. Stiffness (N/m) was characterised by the muscle's ability to resist an external force that modi ed its shape 33 . Elasticity was measured by the logarithmic decrement (log) of the dampened oscillations (dissipation of mechanical energy during one oscillation cycle), thus re ecting the ability of the tissue to recover its shape after being deformed 34 .

Muscle stiffness measurements Field experiments
On the day before the marathon, the rst sample of muscle stiffness was collected. The quadriceps muscle of the thigh and the triceps muscle of the calf were measured. All measurements were performed weekly in a designated room. The participants were supine, on their backs, or on their stomachs on a special bed and they rested for 10 min before muscle stiffness measurements were taken. Testing sites on each muscle were located using a tape measure and marked using a skin-safe pen (Fig. 1). A pillow was placed under the head and a special roller pillow was placed under the lower leg to aid relaxation. One series of three single Myoton measurements of each muscle group (12 points) were measured separately for the left and right leg.

Resting recordings
Immediately after the marathon run, the second sample was collected. All measurements were performed in a designated tent situated past the marathon nish line. Testing sites on each muscle belly were identi ed using a tape measure and marked using a skin-safe pen. A pillow was placed under the head and a special roller pillow was placed under the lower leg to aid relaxation. Again, a series of three single Myoton measurements of each muscle group (12 points) was taken separately for the left and right legs.
Reliability between trials (within session) for one of the selected muscles (two series of 10 single measurements) of each group was tested using the intraclass correlation coe cient (ICC) model.

Statistical analysis
Data were tested for normality using the Shapiro-Wilk test and for homogeneity of variance (Levene's test). Descriptive statistics included the mean, SD, and SE. To compare mean values of the examined variables, repeated measures of one-way ANOVA were used. The independent variable is the time needed to complete the marathon run, whereas the dependent variables were muscle stiffness (MFT, MBS, and hoc Fisher's LSD test. The level of statistical signi cance was set at p = 0.05. Additionally, Cohen's d was calculated, and the effect sizes were determined: 0.35 for small effect size; 0.35 and 0.65 for medium effect size, and 0.65 for large effect size (13). The relationship between the variables was determined using Pearson's product-moment correlation. Statistical power was set to be > 0.90 at the level of p = 0.05. All statistical analyses were made using the STATISTICA ver. 13.1 (StatSoft. Inc., USA) software package.

Results
The 42.195-km run was completed in 4.30,05 h ± 35.12 min., which indicates an intensity of 79.3% ± 7.1% of HRmax. The average body height of the marathon runners was 175.61 ± 5.74 cm, their body weight was 76.17 ± 7.73 kg, and their BMI was 24.44 ± 2.32. A low level of HRmax and VO2max was visible.
Similar relationships can be seen in the case of VO2 at the aerobic threshold (VT1) and anaerobic threshold (VT2). The participants percentage of the Wrocław Marathon on VT1 achieved 76.23% VO2max. In turn, the possibilities at the VT2 threshold were 91.3% and 84.65% HRmax (Table 1). In turn, Fig. 2 shows the course of the variability of speed with the division into individual sections (every 5 km) and the average HRmax on these sections. It can be seen that the beginning of the drop in speed starts at 12 km and continues to the end of the run. Along with the decrease in speed, there was a gradual increase in HRmax which also lasted until the end of the marathon.
The analysis of muscle stiffness levels in relation to their subsequent measurements (before and after the marathon) revealed that a signi cant effect was observed only for the left calf (F = 6.534(1); p = 0.016; medium effect size). No signi cant effect was found for the left quadriceps (F = 2.393(1); p = 0.132), the right calf (F = 3.493(1); p = 0.071), or the right quadriceps (F = 0.876(1); p = 0.357) ( Table 2). The analysis of the Spearman's rank correlation did not reveal any signi cant correlation between running speed or HR level, measured over the entire distance divided into 5 km sections, and muscle stiffness of the quadriceps and triceps calf muscles. On the other hand, signi cant relationships occurred only between the VO2max measurement (measured before the race) and the speed at each of the 5-km sections of the marathon distance and the nish (p = 0.000034 and p = 0.000239, respectively).

Discussion
The aim of this research was to evaluate marathon performance and asses the in uence of this longterm running endurance exercise on the changes of muscle stiffness in middle-aged marathon runners. The hypothesis regarding muscle stiffness was not supported, as the current investigation revealed signi cantly lower levels of stiffness post-marathon for the calf muscles in the left leg (p = 0.016). No signi cant changes were noted with regard to muscle stiffness at the post-marathon assessment in the other two tested muscle groups (quadriceps, in both the left and right lower limbs, and calf in the right limb). Generally, this was very surprising.
The explanation of this phenomenon is likely to be di cult because none of the previously described studies have documented the impact of a prolonged running effort commonly de ned as a marathon on muscle stiffness. Additionally, this requires the consideration of indirect analyses of other variables which effect the marathon effort. This approach is also considered di cult because many of the factors to be analysed were not included in this experiment. The reason for this is that many of these variables are di cult to measure without interfering with the running autonomy. However, it has been well described how long-distance running directly impacts running economy (RE) and muscle damage 11 . It seems reasonable to combine all these factors, due to the non-exclusive relationships, to optimally assess the marathon effort and its direct impact on changes in the runner's body after such a long effort. Knowing this may help runners not only to improve their marathon performance, but also to develop an appropriate training programme, which can optimally prepare them to run 42.195 m, regardless of their level: championship, intermediate, or recreational. An important element of such an analysis is the division into sex, but particularly into age categories, with a special emphasis on 50 + 37 .
Essentially, marathon performance depends on the running economy (RE) in all the word weariness. RE is an 'aerobic demand' to maintain a proper pace: speed over distance. It is de ned as the stationary oxygen uptake (VO2) associated with this speed 18,19 . Comparing our participants with younger marathon runners aged 43.9 ± 8.3, the values of HRmax were lower by an average of 9.2 (bpm) and 4.29 (ml/min/kg) 38 . Larger differences occur in comparison with the group of recreational runners (63 ± 32 km/week) aged 34 ± 8 years. The differences in Hrmax and VO2max are 14.5 (bpm) and VO2max by 18.69 (ml/min/kg), respectively, in favour of the younger runners 39 . Similar relationships can be seen in the case of VO2 at the aerobic threshold (VT1) and anaerobic threshold (VT2). Younger runners are characterised by higher VO2 at these thresholds (3.67 for VT1 and 12.86 for VT2) 39 . Despite the lower values of these indicators, the marathon runners studied in Wrocław consumed more oxygen in relation to their abilities than did younger recreational athletes. HRmax, respectively. Despite this, our marathon runners showed a strong relationship between Vo2 max and speed on each subsequent 5-km section (in the range from 0.000034 to 0.000239). This con rmed previous research, which found that there is a strong relationship between VO2 max and the level of effort in a marathon run.
It is also obvious that the RE must be associated with the marathon runner's individual running technique, and that this, in turn, depends on the resistance of the runner's body to fatigue and falling running speed. In our experiment, marathon runners began to experience a drop in running speed after 12 km of a race. From that moment on, a continuous, slow decrease in speed began, which amounted to approx. 5.6% at the nish line. This did not con rm the reports of Hettinga et al. 40 that during the late stages of the marathon (the last 10-15 km) a considerable deceleration usually occurs. This affects even world-class runners and is recognised by runners as 'hitting the wall' 41 . This is probably due to the fact that our runners are classi ed as slow, recreational runners and they were over 50 years of age, so their marathon effort can be de ned as prolonged (4.30,05 h ± 35.12 min.) but of low intensity.
On the other hand, world-class marathon runners have developed training strategies to manage or prevent fatigue and sharp drops in running speed 42 . The studies by Buckalew et al. 43 and Chan-Roper et al. 44 regarding the effects of fatigue on running technique showed that technique changed by decreases in step length rather than step frequency. These changes were directly responsible for the decreased speed.
Marathon runners are predominantly rear-foot strikers, which is true for both world-class 45 and recreational long-distance runners 46 . This can be applied to our marathon runners with an indication of the activity of the left leg, and with particular emphasis on the triceps muscle of the calf. They noticed a few disadvantages in this matter. A signi cant potential biomechanical limitation of landing with a rearfoot strike pattern is that the foot lands in front of the whole body's centre of mass. This increases the braking force and directly impacts the speed, mainly reducing it by the resulting weaker take-off. This negatively in uences the step length by shortening it. The second disadvantage of the running technique when fatigue appears is that landing almost the whole foot on the ground during the early stance and then continuing during the main amortisation phase signi cantly increases contact time. In turn, the high centre of mass is achieved through knee exion. The greater the knee exion, the longer the foot-ground contact time and the higher the reduction in speed. Additionally, according to Derrick et al. 47 , the presence of fatigue may decrease the utilisation of the stretch shortening mechanism, especially in the hip and knee joints. This causes the knee exors and extensors to tire more quickly, which results in reduced leg stiffness. Despite this assumption, the relationship between running speed on each 5-km stretch (increasing fatigue with each km) and muscle stiffness was not con rmed with no change in muscle stiffness. However, a much greater correlation was found for the triceps muscle of the calf (mean signi cance level: p = 0.354612). This can be con rmed by the fact that this muscle has a greater functional impact in the running step technique. In turn, it weakens the ground reaction forces, thus signi cantly extending the contact time 48 . All these elements mean that there is a signi cant reduction in speed, and the runners thus achieve poor results. In addition, these undesirable factors should be eliminated in training in order to achieve optimal results in the marathon in relation to motor preparation. At the same time, these parameters, which should not weaken the running technique, had a positive effect on muscle stiffness. This did not change after the marathon effort compared to the measurements before the race.
Here another problem arises that was not discussed earlier. It should be assumed that muscle stiffness is partly related to delayed-onset muscle soreness (DOMS). Delayed-onset muscle soreness appears when humans engage in exercise to which they are unaccustomed, or is prolonged in time. It is likely that after a long-term running effort -training for and participating in a run, e.g. a half marathon or marathonrunners show signi cant muscle damage and soreness. Muscle stiffness is associated with delayed muscle soreness. Muscle stiffness is de ned as the change in strength divided by the corresponding change in muscle length. This occurs when the change in muscle length is caused by an external factor, e.g. additional external resistance such as uphill running, or by a change in the external load on the muscle -a sustained effort. In other words, the term 'stiffness' describes the resistance of a muscle to a change in length. Therefore, when the muscle is working (eccentric/concentric contraction, e.g. running step) using its entire length, it is able to generate maximum force, and this should automatically increase its stiffness. On the other hand, when the muscle is working, especially in eccentric contraction, with a signi cantly shorter length (large exion of the lower limb in the knee joint), its length does not change signi cantly, which reduces the generation of force and thus reduces the level of its stiffness. This probably happens in marathon runners.
Following this statement, the question arises of whether, with no changes in muscle stiffness after the marathon, there were changes in DOMS, which are mainly manifested by increased muscle soreness and whether they are two independent or mutually exclusive work-related activities. Changes in the mechanical properties of the muscles observed after prolonged physical activity may be associated with increased joint stiffness. In terms of performance, the increased stiffness was found to be associated with increased speed, increased jump velocity, jump height, and running economy (measured by oxygen consumption) 49 . According to Beck et al. 37 , followed by Kerdok et al. 50 , a critical determinant of running economy is the spring-like storage and return of elastic energy from the leg during a stance.
Here we have to distinguish between two elements -muscle stiffness and joint stiffness -which is often equated with leg spring stiffness. The latter measures the stiffness of the muscle and tendon, but regarding how well a runner is able to recoil the elastic energy generated during ground contact in each stride. Therefore, increases in joint stiffness, mainly by eccentric contraction movement, shorten ground contact 50,51 will generate more elastic energy. In turn, this indicates an improvement in running economy over time and an increase of delayed-onset muscle soreness.
According to Beck et al. 37 , it can be concluded that the assessment of older runners may be indirectly based on leg stiffness, through reduced tendon stiffness 52,53 , lower active peak ground vertical reaction forces (GRF) 51 , and greater exion at the knee joint at landing 54,55 . This suggests that leg spring stiffness decreases with age 37 . Did this occur in our marathon runners? Although DOMS was not the subject of the study and we do not have relevant data, it should be assumed that the DOMS was at the tolerance level of the runner's body immediately after the end of the run. The question therefore is how would the DOMS and muscles stiffness behave if measurements were taken several times, e.g. 12 h, 24 h, or 48 h after the marathon? These unknowns were partially answered by Chleboun et al. 56 , who stated that muscle swelling, as one of the factors of DOMS, does not necessarily account for the sudden increase in postexercise stiffness, whatever the determinant of the subsequent muscle stiffness may be.
This is one of the limitations of this study: the absence of subsequent measurements of muscle stiffness, e.g. 12 h or 24 h after completing the marathon. This was not due to the technical feasibility of the measurements, but to the personal reasons of the competitors. Such measurements would have also allowed us to observe the changes in DOMS in relation to the delayed changes. Another limitation is the lack of a running technique evaluation on video recording e.g. 15 km or 40 km into the race. This would have allowed us to correctly describe the marathoners' running technique and juxtapose it with VO2 in order to evaluate their running economy.

Conclusion
The long-term, low-intensity running effort (marathon) in older recreational runners, along with a low level of HRmax and VO2max, showed no statistically signi cant changes in muscle stiffness (quadriceps and calf muscles). There was, however, reduced muscle stiffness, but only in the triceps calf of the dominant (left) leg. Additionally, when we consider the failure to keep an optimal running economy, expressed as a technical disorder (shortened running step, increased ground contact time, lowering the legs in the knee joint) along with increasing fatigue, we can surmise why muscle stiffness did not change post-exercise. Therefore, the hypothesis that muscle mechanical properties and resting tone may change after prolonged exercise was not con rmed in this experiment. In turn, we can rather con rm that this sustained effort may affect the DOMS level by causing an increase in post-exercise muscle soreness.
Moreover, in order to optimally evaluate the marathon run and prepare an adequate training programme, we need to consider a direct and indirect analysis of all the above-mentioned variables, such as the running economy, running technique, HRmax and VO2max and DOMS. These variables signi cantly affect the marathon effort. Figure 1 Arrangement of the measurement points of the quadriceps muscle of the thigh and the triceps muscle of the calf