Low markers of muscle damage and inflammation following a 3-day trail run

Objectives . To investigate the effect of a 3-day trail run on markers of muscle damage and inflammation in recreational runners. Main outcome measures . Pre-and post-stage and 24-hour and 72- hour post-race concentrations of serum creatine phosphokinase (CPK), high sensitivity C-reactive Protein (hsCRP), cortisol, cardiac Troponin T (cTnT), and osmolality (sOsm) as well as urinary myoglobin (uMb), changes in body mass, delayed onset muscle soreness (DOMS) and thigh circumference (TC) were measured. Continuous recordings of heart rate (HR) and intestinal temperature (Tintest ) were made throughout each stage. Results . Heart rate ranged between 77% and 83% age-predicted maximum (APmax) and Tintest between 36.1 and 40.2oC during the three stages. Significant rises in mean serum CPK, hsCRP, sOsm and blood neutrophil count reached peak concentrations of 1488U/l, 8.91mg/l, 298mosm/l and 10.21 109/l (p 2.0%.


Introduction
Trail running events are becoming increasingly popular with amateur athletes. 1These are generally regarded as more strenuous than road running due to the nature of the trails, which can involve diverse challenges including single track paths on steep ascends and descends in mountains, crossing rivers and running along grasslands and through forests. 2 Although physiological response to single-day trail running has been assessed, [1][2][3][4] the cumulative effects of multi-day trail running on markers of muscle damage and inflammation have not yet been reported.Prolonged endurance exercise causes muscle damage that initiates an inflammatory response and subsequent remodelling of muscle. 5The extent of this damage is augmented by increases in exercise intensity, the eccentric component of contraction, [6][7][8] heat stress index and dehydration. 3The greater contractile load per unit in muscles of the lower limb, as they contract eccentrically during downhill running, 8 has been associated with increased mechanical damage to the muscle fibres, resulting in muscle membrane leakage and elevated concentrations of circulating muscle enzymes and proteins. 9Systemic markers of inflammation also rise 5,7 and swelling, decreased mobility and delayed-onset muscle soreness (DOMS) are common. 5,6The presence of myoglobin in the urine has been reported in severe cases.
Although the direct cause-and-effect relationship between dehydration and hyperthermia is currently contentious, 10 it has been reported that these augment exercise-induced muscle damage, 3,4   detrimentally affect performance and pacing during trail running and increase post-exercise DOMS. 3,4,11Cleary et al. 11 reported an association between dehydration and hyperthermia and attributed an increase in muscle damage to the increased degradation of muscle proteins with elevated deep-muscle temperature.
The aims of the study were therefore to determine effects of a multiday trail run on the markers of muscle damage and inflammation in experienced recreational runners, measuring serum and urinary levels of selected skeletal muscle, cardiac and hepatic proteins in association with changes in red and white blood cell and serum cortisol concentrations before and after every stage and at 24 hours post-race (24PR) and 72 hours postrace (72PR).A further aim was to assess the possible effect of dehydration and hyperthermia on the markers of muscle damage and inflammation.
It was hypothesised that the three consecutive days of trail running would result in elevations of systemic and urinary markers of skeletal muscle damage and inflammation that are higher than previously reported during road running events of similar duration, and that the muscle damage and inflammation would be augmented by hyperthermia and dehydration.and did not use chemical stimulants, were accepted into the study.Nineteen (6 males, 13 females) completed all three stages of the race and 15 runners (4 males, 11 females) completed all within-and postrace assessments.

Setting
The Three Cranes trail run, over 3 days and a total distance of 95 km, was divided into 3 consecutive stages comprising 29.3, 37.9 and 27.8 km, starting and finishing each day at the same base camp.Athletes were accommodated in a race village and full catering was provided for the duration of the race, including at the aid stations along the route.The routes consisted of gravel and forestry roads, narrow rocky mountain footpaths and grassy jeep track.Elevation gains reached 1 020, 1 226 and 680 m, while elevation losses were recorded at 1 021, 1 231 and 687 m during S1 (Stage 1), S2 (Stage 2) and S3 (Stage 3) respectively (Table 1).Selected images of the running terrain are presented in Fig. 1.

Baseline measurements
Following race registration the afternoon before the race, basic anthropometric measurements were recorded, including body mass (kg), stature (cm) in bathing suits without shoes, thigh circumference (TC) (measured 15 cm above the superior border of the patella) and four-site skinfold (supra-iliac, subscapular, biceps and triceps) for the determination of % body fat. 12A pre-race questionnaire detailing the athletes' running and racing experience, training terrain and health status was also completed.

Pre stage
The subjects presented themselves to a designated testing area 30 -90 minutes before the start of the stage, handing in a first earlymorning urine sample.TC was measured, venous blood sampling was conducted in the seated position and resting heart rate (HR) and blood pressure (BP) were recorded after a 3 -5-minute period of relaxation.A simple pre-stage questionnaire including a rating of the degree of muscle soreness they were experiencing, was completed and the subjects were asked to keep a record of their fluid intake and urine output during the stage.After breakfast and final voiding of bladders, body mass (measured in running attire without shoes), was taken within 5 minutes prior to the start of the event.

Within stage
Environmental conditions and temperature were supplied on the hour by a meteorological station located 9.5 km from the base camp.Heart rate was recorded using a polar HR monitor (Polar Electro OY, Finland) at 5-minute intervals and % age-predicted maximum (APmax) was determined according to the formula, 220-age.

14
A subsample of 12 athletes volunteered to ingest the Cor-Temp disposable tablets, containing temperature sensors (HQ Inc, Palmetto, FL), at least 3 hours prior to the start of each stage.The HR and intestinal temperature (T intest ) data are part of a more detailed study focussing on the relationship between T intest , HR and hydration status.

Post stage
The subjects proceeded directly to the designated testing area where BP, mass and TC were measured within 3 -5 minutes, blood and urine samples were taken and a short DOMS and post-stage questionnaire providing details regarding the use of non-steroidal anti-inflammatory drugs (NSAIDs) and muscle soreness, were completed.In available athletes (n=10), a further measurement of TC was taken 4 hours after completion of S1 and S2.
The same protocol was followed pre-and post-stage on the 3 days of the race.

Post race
At 24PR and 72PR, participants presented for further blood/urine sampling, BP, HR and anthropometric measurements.They were also requested to complete a DOMS questionnaire for the 5 days following the race, using a five-point Likert scale, and to return this together with a general post-race questionnaire, following completion of the study.

Haematological analysis and anthropometric measurements
Each measurement was carried out by the same researcher for all subjects and at each time point.Venous blood samples were drawn from the antecubital fossa, with subjects in the seated position, within 5 -15 minutes of completing the stage.Blood samples for the assessment of full blood count (FBC) and serum osmolality (sOsm) and urine samples were stored at 4ºC and transported to a commercial pathology laboratory.Complete blood counts were measured on an Advia-120 Hematology Analyzer (Siemens Healthcare Diagnostics, Deerfield, IL) and included erythrocyte indices and differential leukocyte counts.Both urine and serum osmolality were measured by freezing-point depression, using a Kyoto Daiichi osmostat, OM 6020 (Japan).Urine samples were also assessed for myoglobin (uMb) and specific gravity using the refractive index method on a Beyer Test Strip.
Further aliquots of serum, separated by centrifugation @ 3 000 rpm and stored in dry ice were transferred to an -80ºC ultrafreezer or transported to a commercial pathology laboratory for analysis of creatine phosphokinase (CPK), cortisol, cardiac troponin T (cTnT) and high sensitivity C-reactive protein (hsCRP) concentrations.

Statistical analyses
Data are presented as mean ± standard deviation (SD).The significance of the accumulative time-dependent stage-induced changes from pre-race (S1 pre ) to post race (S1 post , S2 pos t, S3 post ), as well as recovery rates were assessed comparing S2 pre , S3 pre , 24PR and 72PR to baseline (S1 pre ,), S2 pre and S3 pre were assessed for the entire group using repeated measures one way analysis of variance.The time point of the significant differences was confirmed using a Tukey post hoc analysis.
Comparisons between NSAID users and non-users were conducted using independent Student's t-tests.Pearson's product moment coefficient of correlation, with a confidence interval (CI) of 95%, was used to test the relationship between the changes in measured outcomes including CPK, neutrophil concentrations, hsCRP and serum cortisol.
All statistical calculations were performed using SPSS, version 18 (SPSS Inc., Chicago, USA).Level of significance was set at p<0.05.

Environmental conditions
Temperature recorded on the hour during the three stages of the race ranged from 11.5ºC to 22.8ºC (Table 1).It did not rain, maximum wind speed recorded was 2.8 m/s and the relative humidity ranged from 54% to 97%.

Subjects
As is shown in Tables 2 and 3, athletes ranged from 25 to 50 years of age, their weekly training distance averaged 65.9±20.1kmper week for 12.4 years (range 2 -27 years) and they presented without abnormalities in their vital signs.Of the 19 subjects, 12 used NSAIDs, including aspirin, ibuprofen and diclofenac.Of the 21 subjects who initially agreed to participate in the study, one subject (male) withdrew after S1 due to an ankle injury and another (female) after S2 due to medical reasons.The baseline physical characteristics of the remaining 19 subjects are provided in Table 2. Four subjects were however unable to provide blood samples at 24PR and 72PR.

Intensity of effort
The mean ±SD and range of time spent completing each stage and average HR on the run, are given in Table 3.Total average running time of the athletes was 12h57±2h51.

Markers of muscle damage and inflammation
As shown in Table 4, these included a significant increase in circulating neutrophil concentrations (p<0.001) which peaked at 10.21±1.54 10 9 /l at S1 post , serum CPK and hsCRP which peaked at S3 post at 1 488± 1 053U/l (p≤0.001) and 8.91±6.63mg/l(p≤0.001),respectively.cTnT and uMb were undetected in all samples throughout the 3-day event.
An exercise-induced increase in serum cortisol concentration was only detected following S2 post .TC decreased significantly from 54.1±4.4 cm at S1 pre to 51.8±3.9cm at S1 post (p<0.001) and returned to the pre-race measurement of 54.1±4.0 cm at 24PR.DOMS ranged from 4.8±1.6,5.6±1.8 and 5.1±1.1 at S1 post , S2 post and S3 post , respectively, and decreased to 1.73±1.3
The pooled data examining the relationship between the change of sOsm and change in serum CPK for the three stages (n=57) revealed an insignificant positive correlation (r=0.034,95% CI [-0.228, 0.291]. The maximum T intest ranged between 38.3ºC and 40.2ºC and only exceeded 40º C in two of the 12 athletes monitored (Table 5).The relationship between change in T intest and serum CPK was insignificant (p>0.05) for the 11 individuals from whom complete sets of data were available (r=0.24, 95% CI [-0.42, 0.734]).

Users of NSAIDs
The 12 athletes who used NSAIDs had maximum serum CPK and hsCRP concentrations of 1 332±943.5 U/l and 8.58±6.7 mg/l at S3 post and the non-users 1 754±1 251.3 U/l and 9.47±7.0mg/l, with no significant difference between the groups (p=0.456;0.788).The neutrophil count reached a maximum of 9.95±2.1 and 9.75±0.4 10 9 /l, respectively, for users and non-users (p=0.82).There was also no significant difference between NSAID users and non-users in terms of serum cortisol, post race DOMS scores, running times, TC or sOsm (p>0.05).

Evidence of muscle damage and inflammation
The results of the present study indicate that very little muscle damage and inflammation occurred during 3 days of trail running despite athletes running for a total average of 12h57 at an average HR of 77 -83% APmax (Table 3).The serum CPK concentration, which increased progressively to reach peak concentrations at S3 post , indicated only a mild cumulative effect of muscle damage during the race, which rejects the original hypothesis.Furthermore, the changes in neutrophil count, serum cortisol and hsCRP concentrations and DOMS also confirm low levels of inflammation and a rapid recovery.Most athletes in our study had no muscle soreness at 72PR, which correlated with the CPK concentration that had dropped close to the clinical upper limit of normal by 72PR. 15The consistently low release of muscle proteins into the bloodstream in all 19 subjects, which was also not accompanied by elevation in cTnT and uMb in this study, confirms a profile of low degrees of muscle damage.Further evidence is the fact that TC was not significantly elevated at any post-stage or post-race time-point, but was reduced after S1 (p<0.001),confirming previous findings of reduced swelling and a post-race decrease in muscle mass.16   The low systemic markers of muscle damage and inflammation, when compared with previous findings following the Comrades Marathon 16 confirm the findings of Millet et al. 1 who, in their study on the neuromuscular consequences of extreme running in a 166 km mountain ultra-marathon, reported that post-race serum concentrations of CPK, hsCRP and neutrophils were lower than those measured after a road race with similar finishing times. 1 These researchers attributed their findings of low concentrations of systemic markers of muscle damage and inflammation to the relatively soft underfoot surfaces and to the athletes frequently being forced to walk, jump and climb due to the technical demands of the terrain.
During extensive exercise-induced muscle damage myoglobin may be released into the urine and be indicative of exertional rhabdomyolysis and possible risk of renal failure. 8Clarkson 9 however reported that exertional muscle damage in healthy athletes can cause profound serum CPK elevations without renal impairment.In our study the absence of uMb was confirmed by the relatively low increases in systemic neutrophil, serum CPK and hsCRP concentrations.
In this study we suspect that although the primary factor which reduced the amount of repetitive and eccentric unidirectional stress encountered during the race was most probably the underfoot surfaces, the majority of which were primarily soft, large fluctuations in the pace of running and varied muscle recruitment patterns over the different terrains may also have played a role.
The positive correlation between DOMS scores and CPK concentrations supports the findings of Nieman et al. 2 who, in their study on 60 participants in the 160 km 1-day Western States Endurance Trail Run in the Sierra Nevada Mountains in northern   California, showed that there were significant associations between CPK, muscle soreness and the cytokines, interleukin (IL)-6, IL-10, IL-1ra (receptor antagonist), granulocyte colony-stimulating factor and macrophage inflammatory protein 1β.

Systemic markers of cardiac damage
The effect of prolonged strenuous exercise on systemic cardiac markers of damage has been studied extensively, [18][19][20] with evidence of transient elevations during and immediately after exercise, which return to normal within 3 days in healthy athletes. 18,19These temporary elevations have been hypothesised to be due to myocardial stress and reversible cardiomyocyte membrane damage. 18,19Exercise is known to cause an increased myocardial oxygen demand and cardiac troponin turnover in all athletes, 18 which might be linked to tachyarrhythmias and sudden cardiac death, when associated with prolonged increases (>3 days) in cTnT concentrations above 0.05µg/l. 18At no stage during our study were increased cTnT concentrations measured, supporting the attenuated increase in serum CPK concentration and absent uMb values as well as the lower concentration of serum cortisol despite maintenance of an intensity of effort which fluctuated from 63 to 112% APmax.It is possible that serum cTnT also did not increase due to the variation in HR (60 -220bpm) that occurred during this race, which may have stimulated the cardiac muscle at irregular intervals and possibly reduced myocardial stress by permitting periods of recovery.

Users of NSAIDs
Both NSAID users and non-users were included in this study following recent findings that although markers of muscle inflammation are changed by NSAID usage, degree of muscle damage is unaffected.

21,22
Nieman et al. 2 reported that NSAID users did not have reduced race times, muscle damage or DOMS, while Friden and Lieber 6 reported that administration of NSAIDs after eccentric exercise resulted in a short-term benefit of pain relief, but a long-term detrimental effect on muscle adaptation, inhibiting protein synthesis by suppressing the inflammatory reaction.Paulsen et al. 22

also indicated that although
NSAIDs inhibited prostaglandin synthesis and local and systemic responses, they did not affect actual markers of muscle damage.In this study there was however no statistical difference in the measured markers of muscle damage or inflammatory response between NSAID users and non-users.

Dehydration, intestinal temperature (T intest ), HR and evidence of muscle damage
Although some athletes in our study experienced up to 4% body mass loss and others, on occasion, raced at a HR of more than 100% APmax (Table 5), these athletes did not present with clinical signs of dehydration, severe hyperthermia or increased muscle damage as reflected by changes in sOsm, T intest >40ºC or changes in serum CPK concentration, respectively.As the statistically significant (p<0.05)inverse correlation between % change in body mass and post-pre change in sOsm was low (r=-0.365),sOsm, widely reported golden marker of hydration status, 23 was used to quantitate changes in hydration status.
The correlation between hydration status and systemic markers of muscle damage, as reflected by stage-induced changes in sOsm and serum CPK concentrations, although statistically significant, was low.Hence it cannot be concluded from the 51 sets of paired data reported in this study that hydration status has an overriding effect on systemic markers of muscle damage.
In the 12 individuals in whom continuous recordings of T intest were recorded (Table 5), the correlation between race-induced changes in T intest and systemic markers of muscle damage was also low and statistically insignificant.The data provided in this study, although

Conclusion
The relatively low post-race concentrations of systemic and urinary markers of muscle damage and inflammation, 5 when compared with those reported following road running events of similar duration, 15 are attributed to softer underfoot surfaces, large fluctuations in pace of running and varied muscle recruitment patterns over the widely differing terrains. 1The sporadic increases in intensity of effort, rises in T intest , substantial body mass loss and increases in serum osmolality during the event, did not confirm previous suggestions 3,4,11 that thermal and hydration status is directly related to the degree of muscle damage.
It would be of interest to the investigate the impact of pre-race preparation on markers of muscle damage and inflammatory response found following this multi-day trail running event and to control the nutritional and fluid intake in future field work on multiday trail running.

Table 5 . Individual T intest , and associated HR, changes in hydration status and peak serum CPK concentration (n=12)
maximum heart rate based on a relatively small sample size, do not provide any support for the suggestion that rises in core body temperature exaxerbate muscle damage.