SAJSM vol 24 No. 1 2012 15 original research Introduction Trail running events are becoming increasingly popular with amateur athletes.1 These 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,1-4 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.5 The extent of this damage is augmented by increases in exercise intensity, the eccentric component of contraction,6-8 heat stress index and dehydration.3 The 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.9 Systemic markers of inflammation also rise5,7 and swelling, decreased mobility and delayed-onset muscle soreness (DOMS) are common.5,6 The presence of myoglobin in the urine has been reported in severe cases.5 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.3,4,11 Cleary 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 post- race (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. Method Ethical clearance This 8-day observational cohort study took place during a 3-day trail run and for 5 days following completion of the Three Cranes Trail Run, at Karkloof, KwaZulu-Natal, South Africa on 25 - 27 February 2011.  Following approval by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal, subjects gave written consent after having been informed of the experimental procedures.    Subjects Twenty-one apparently healthy subjects, who met the inclusion criteria (age: ≤50 and an average training distance of 60 km per week) Abstract 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<0.001), respectively. No evidence of elevations in uMb and cTnT were detected. The stage-induced increments in DOMS correlated positively with CPK, r=0.71; 95% CI [0.62, 0.78], TC decreased significantly post S1post and S2post (p<0.05) and a maximum mean body mass loss of 3.09% (±1.04%) occurred during S2. Conclusion. Three consecutive days of 95-km trail running resulted in low markers of muscle damage and inflammation, despite the maintenance of a heart rate above 77% APmax, Tintest rising above 39oC and mean body mass decrement of >2.0%. Low markers of muscle damage and inflammation following a 3-day trail run Emmerentia C Denissen (MTech Hom) Anton H de Waard (MTechHom) Navin R Singh (MSc (Eng)) Edith M Peters (PhD) Division of Human Physiology, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu- Natal, Westville, Durban, South Africa Correspondence to: Edith Peters-Futre (futree@ukzn.ac.za) 16 SAJSM vol 24 No. 1 2012 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 post- race 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 Fig. 1. Selected images of the running terrain. SAJSM vol 24 No. 1 2012 17 determination of % body fat.12 A pre-race questionnaire detailing the athletes’ running and racing experience, training terrain and health status was also completed. Daily protocol Pre stage The subjects presented themselves to a designated testing area 30 - 90 minutes before the start of the stage, handing in a first early- morning 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 (Tintest) data are part of a more detailed study focussing on the relationship between Tintest, HR and hydration status. 13 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 dr y ice were transferred to an -80ºC ultrafreezer or transported to a commercial pathology laboratory for analysis of creatine phosphokinase (CPK), cortisol, cardiac trop onin 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 (S1pre) to post race (S1post, S2post, S3post), as well as recovery rates were assessed comparing S2pre, S3pre, 24PR and 72PR to baseline (S1pre,), S2pre and S3pre 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 co- efficient 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.  Results 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%. Table 2. Mean ± SD baseline physical characteristics of subjects (n=19) Variable Mean±SD Age (years) 39.3±7 Height (cm) 169.0±10 Mass (kg) 65.8±12 % body fat 21.7±4 Resting heart rate (bpm) 56.9±5 Systolic blood pressure (mmHg) 124.7±7 Diastolic blood pressure(mmHg) 81.8±7 Table 1. Elevation changes (m) and ambient temperature rang- es during the three stages of the trail run Elevation gain  Elevation loss Ambient temp- erature range (ºC) Day 1 1 020 1 021 11.5 - 21.7 Day 2 1 226 1 231 12.4 - 22.8 Day 3 680 678 12.1 - 21.2 Total 2 926 2 930 18 SAJSM vol 24 No. 1 2012 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±20.1km per 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±1.54 109/l at S1post, serum CPK and hsCRP which peaked at S3post 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 S2post. TC decreased significantly from 54.1±4.4 cm at S1pre to 51.8±3.9cm at S1post (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 S1post, S2post and S3post, respectively, and decreased to 1.73±1.3 at 24PR. Significant positive correlations were evident between blood neutrophil concentrations and serum CPK, r=0.27, 95% CI [0.11, 0.41], serum CPK and hsCRP concentrations, r=0.50, 95% CI [0.29, 0.66] and DOMS and CPK, r=0.71, 95% CI [0.62, 0.78]. Dehydration, intestinal temperature (Tintest), HR and muscle damage The mean % body mass loss for the entire group (n=19) during the three stages was 2.9±0.7, 3.1±0.8 and 1.9±0.9, while the mean sOsm (n=19) increased from 288.9±4.8 to 293.7±5.7 (p=0.003), 288.4±6.4 to 295.6±6.0 (p=0.003) and 292.2±4.1.to 295.0±5.6 (p=0.006) mOsm/ kg, during S1, S2 and S3, respectively. When the pooled data for each stage were compared (n=51), the paired post-pre changes in sOsm correlated inversely with the changes in % body mass, r=-0.36, 95% CI [-0.57,-0.094]. 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 Tintest 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 Tintest 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 S3post and the non-users 1 754±1 251.3 U/l and 9.47±7.0 mg/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±0.4 109/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). Discussion 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 S3post, 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.15 The consistently low release of muscle proteins into the bloodstream in all 19 subjects, which was also not accompanied Table 3. Training status and performance characteristics of athletes (n=19) Characteristics Mean±SD Range Running experience Number of years Number of competitive endurance events 12.4±8.1 136.3±55.6 2 - 27 18 - 500 Weekly training distance (kilometres per week) 65.9±20.1 12.5 - 105 Number of days per week on different training terrains Hills Off road incl. forest /trail/beach Road 1.4±0.8 1.7±1.5 3.9±1.3 1 - 4 0 - 6 0 - 5 Race time (hour:minute:second) Stage 1 Stage 2 Stage 3 4:04:31±25:54 5:39:12±25:31 3:14:15±21:06 3:06:06 - 5:22:48 4:14:56 - 7:46:27 2:38:38 - 6:51:50 Average heart rate (beats per minute) Stage 1 Stage 2 Stage 3 150.8±21.3 140.7±22.5 138.5±23.3 73 - 191 60 - 186 75 - 198 Mean as %APmax* Stage 1 Stage 2 Stage 3 83±8.8 78±7.8 77±8.1 71 - 112 55 - 105 63 - 105 Data presented as mean (±SD) and range. *Age-predicted maximum (220-age). SAJSM vol 24 No. 1 2012 19 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 Marathon16 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.8 Clarkson9 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 Table 4. Mean±SD white and red blood cell indices and markers of muscle damage and inflammatory response before and after every stage and at 24PR and 72PR Variable Stage 1 Stage 2 Stage 3 24PR 72PRPre Post Pre Post Pre Post Red blood cells (10 12 /l) 4.7 ±0.5 4.7 ±0.5 4.5 ±0.4 4.6 ±0.5 4.4 ±0.4 4.4 ±0.4 4.1 ±0.4 4.3 ±0.4 Haemoglobin (g/dl) 14.4 ±1.3 14.3 ±1.2 13.7 ±1.0 14.1 ±1.2 13.2 ±0.9 13.3 ±1.1 12.7 ±0.9 13.2 ±1.2 Haematocrit (%) 42.4 ±3.9 42.1 ±3.3 40.2 ±2.9 41.1 ±3.2 39.9 ±2.7 39.5 ±2.9 38.1 ±2.5 40.5 ±3.4 White blood cells (10 9 /l) 6.0 1.2 12.8* ±1.7 6.5 ±1.3 12.6** ±2.0 7.1 ±1.3 9.7*** ±2.2 6.7 ±1.2 6.5 ±1.5 Neutrophils (10 9 /l) 2.9 ±0.7 10.2* ±1.5 3.4 ±1.0 9.7** ±1.9 3.7 ±1.0 7.3*** ±2.1 3.6 ±1.0 4.2 ±1.4 Lymphocytes (10 9 /l) 2.1 ±0.5 1.4 ±0.5 2.2 ±0.6 1.73 ±0.6 2.4 ±0.7 1.5 ±0.4 2.1 ±0.6 1.6 ±0.5 Eosinophils (10 9 /l) 0.3 ±0.2 0.1 ±0.1 0.2 ±0.2 0.1 ±0.1 0.3 ±0.2 0.1 ±0.1 0.2 ±0.2 0.1 ±0.1 Basophils (10 9 /l) 0.04 ±0.02 0.1 ±0.03 0.0 ±0.02 0.06 ±0.05 0.05 ±0.02 0.0 ±0.02 0.04 ±0.02 0.03 ±0.01 CPK (U/l) 116.5 ±54.6 275.4* ±105.9 419.8 ±212.6 971.6** ±534.2 953.7 ±579.3 1488*** ±1053 595.6* ±361.4 201.9* ±111.3 hsCRP (mg/l) 0.7 ±0.5 - - - - 8.9* ±6.6 6.6* ±6.2 2.0* ±1.7 Cortisol (nmol/l) 759.1 ±154.8 779.1 ±233.3 729.2 ±134.1 934.9** ±216.9 646.8 ±112.4 583.2 ±213.2 - - uMb (mcg/ml) n/d n/d n/d n/d n/d n/d n/d n/d cTnT (µg/l) - - - - <0.01 <0.01 <0.01 - Thigh circum (cm) 54.1 ±4.4 51.8* ±3.9 54.1 ±4.6 53.3 ±4.6 53.4 ±4.4 53.8 ±4.2 54.1 ±3.9 53.5 ±3.8 *v. S1pre, p<0.001 **v. S2 pre, p<0.001 ***v. S3 pre, p<0.001 uMb=urinary myoglobin; cTnT=cardiac troponin T; n/d=not detected. 20 SAJSM vol 24 No. 1 2012 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,18-20 with evidence of transient elevations during and immediately after exercise, which return to normal within 3 days in healthy athletes.18,19 These temporary elevations have been hypothesised to be due to myocardial stress and reversible cardiomyocyte membrane damage.18,19 Exercise 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.18 At 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 Lieber6 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 (Tintest), 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, Tintest >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 Tintest were recorded (Table 5), the correlation between race-induced changes in Tintest and systemic markers of muscle damage was also low and statistically insignificant. The data provided in this study, although Table 5. Individual Tintest , and associated HR, changes in hydration status and peak serum CPK concentration (n=12) Subject Number Max Tintest Min Tintest Mean Tintest Max HR (bpm) Mean HR Max% APmax HR* Mean% APmax HR * ∆sOsm (mOsm/kg) ∆Body Mass % Max serum CPK ( U/l) 1 39.2 36.6 38.5 176 167 100 95 -3 -3.4 # 2 38.7 37.2 38.3 161 148 94 87 -6 -2.9 772 3 39.4 37.3 38.7 164 153 88 82 13 -2.7 1562 4 39.6 36.8 38.7 193 144 112 83 -6 -4.1 1057 5 39.2 35.2 37.4 168 154 95 87 10 -2.8 1523 6 39.8 36.1 37.7 168 160 92 87 7 -3.1 4478 7 38.7 36.9 38.2 152 140 89 82 11 -3.0 772 8 39.2 37.3 38.1 180 159 95 72 -23 -2.3 1198 9 40.2 37.1 38.0 181 144 102 81 -5 -3.3 1076 10 38.9 37.4 38.4 156 150 84 81 5 -3.2 1089 11 38.3 35.8 37.4 146 135 84 78 6 -2.8 1057 12 40.1 37.3 38.5 171 149 90 79 7 -2.3 1151 Mean 39.3 36.8 38.2 168 150.3 93.8 82.8 4.17 2.99 1430 SD 0.6 0.7 0.4 13.4 9.0 8.0 5.8 9.42 0.49 1042 Tintest=intestinal temperature; max=maximum; min=minimum; HR=heart rate; bpm=beats per minute; SD=standard deviation; CPK=creatine phosphokinase.* age-predicted maximum heart rate 14 # subject withdrew after completing S1. SAJSM vol 24 No. 1 2012 21 based on a relatively small sample size, do not provide any support for the suggestion that rises in core body temperature exaxerbate muscle damage. 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.1 The sporadic increases in intensity of effort, rises in Tintest, substantial body mass loss and increases in serum osmolality during the event, did not confirm previous suggestions3,4,11 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. Acknowledgements Special thanks are extended to: • Ms Heidi Mocke and the Wildlands Conservation Trust for permitting the collection of these data at the 2011 Three Cranes Challenge multiday trail run and for providing the research team with accommodation in the ‘race village’ located in the Bushwillow Campsite. They are also thanked for provision of the topographical data re the altitude gains and deficits during the race. • Ergonomics Technologies, Pretoria and the MRC Research Unit for Exercise Science and Sports Medicine for the loan of Cor Temp data recorders which made the continuous monitoring of HR and Tintest possible. • Professor AJ McKune and Mr D Naicker for their support with the field side collection of data reported in this paper. • Ampath Laboratories for its assistance with phlebotomy and chemical pathology. • The SA Weather Office for provision of precise data regarding the environmental conditions during the different stages of the race. • Asokaran Rajh for assistance with graphic artwork. REfERENCES 1. Millet GY, Tomazin K, Verges S, et al. Neuromuscular consequences of an extreme mountain ultra-Marathon. PLoS One 2011;6(2):e17059. 2. Nieman DC, Dumke CL, Henson DA, McAnulty SR, Gross SJ, Lind RH. Muscle damage is linked to cytokine changes following a 160-km race. 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