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PLOS One logoLink to PLOS One
. 2020 Sep 23;15(9):e0239158. doi: 10.1371/journal.pone.0239158

Biochemical markers after the Norseman Extreme Triathlon

Christoffer Nyborg 1,2,*, Jørgen Melau 1,2,3, Martin Bonnevie-Svendsen 2, Maria Mathiasen 4, Helene Støle Melsom 1,2, Andreas B Storsve 5, Jonny Hisdal 1,2
Editor: Pedro Tauler6
PMCID: PMC7510962  PMID: 32966338

Abstract

Prolonged exercise is known to cause changes in common biomarkers. Occasionally, competition athletes need medical assistance and hospitalisation during prolonged exercise events. To aid clinicians treating patients and medical teams in such events we have studied common biomarkers after at The Norseman Xtreme Triathlon (Norseman), an Ironman distance triathlon with an accumulated climb of 5200 m, and an Olympic triathlon for comparison. Blood samples were collected before, immediately after, and the day following the Norseman Xtreme Triatlon (n = 98) and Oslo Olympic Triathlon (n = 15). Increased levels of clinical significance were seen at the finish line of the Norseman in white blood cells count (WBC) (14.2 [13.5–14.9] 109/L, p < 0.001), creatinine kinase (CK) (2450 [1620–3950] U/L, p < 0.001) and NT-proBNP (576 [331–856] ng/L, p < 0.001). The following day there were clinically significant changes in CRP (39 [27–56] mg/L, p < 0.001) and Aspartate Aminotransferase (AST) (142 [99–191] U/L, p < 0.001). In comparison, after the Olympic triathlon distance, there were statistically significant, but less clinically important, changes in WBC (7.8 [6.7–9.6] 109/L, p < 0.001), CK (303 [182–393] U/L, p < 0.001) and NT-proBNP (77 [49–88] ng/L, p < 0.01) immediately after the race, and in CRP (2 [1–3] mg/L, p < 0.001) and AST (31 [26–41] U/L, p < 0.01) the following day. Subclinical changes were also observed in Hemoglobin, Thrombocytes, K+, Ca2+, Mg2+, Creatinine, Alanine Aminotransferase and Thyroxine after the Norseman. In conclusion, there were significant changes in biomarkers used in a clinical setting after the Norseman. Of largest clinical importance were clinically significant increased WBC, CRP, AST, CK and NT-proBNP after the Norseman. This is important to be aware of when athletes engaging in prolonged exercise events receive medical assistance or are hospitalised.

Introduction

There is a growing interest in ultra-triathlons competitions worldwide [1, 2]. Occasionally, competition athletes need medical assistance and hospitalisation due to fatigue, illness or accidents [3]. Prolonged exercise is known to cause changes in common biomarkers [4–6]. There are major changes in serval biomarkers after Ironman distance triathlons [7–9]. Knowledge about what range of change can be considered “normal” regarding the most common biomarkers used in clinical diagnostics is therefore warranted, as this will allow for the findings in patients who have participated in these types of competitions to be evaluated [10, 11]. Our aim is to publish a reference material for medical teams working at The Norseman Xtreme Triathlon (Norseman) and other Ironman triathlons.

Norseman has been rated as one of the world’s toughest triathlons by popular media [12]. It is probably one of the most popular Xtreme Triathlon Competition in the world, with more than 5000 applicants for the start slots in the 2019 race. It is also the current host of the World Championship in triathlon. The competition is an Ironman distance triathlon that takes place in Norway [13]. It starts with a jump from a ferry and a 3800 meter swim in the Hardanger Fjord. Then the athletes race 180 km by bicycle with an elevation of more than 3000 meters, before finishing off with a 42.2 km run to Gaustatoppen. The total elevation of the race course is 5200 m.

We examined Creatinine Kinase (CK), N-terminal pro Brain Natriuretic Peptide (NT-proBNP), Creatinine, C-reactive protein (CRP), White blood cells (WBC), Thrombocytes, Hemoglobin (Hb), Sodium (Na+), Potassium (K+), Calcium (Ca2+), Magnesium (Mg2+), Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH), covering heart function, inflammation markers, markers of cell injury, electrolytes and thyroid function [4–9, 14–20]. For comparison of the impact of different types of triathlons we have also examined the same biomarkers after an Olympic triathlon. Our hypothesis where that there would be greater changes I the biomarkers after Norseman compared to after an Olympic triathlon.

Materials and methods

Study population

In the present study, volunteers were recruited from among participants in the Norseman races of 2016, 2018 and 2019, and from the Oslo Triathlon (Olympic distance) in 2018. Norseman and Oslo Triathlon are both contested in Norway in August. All participants received an e-mail 1–2 months prior to the races with information and the possibility to sign up for the study. All volunteers that completed the respective races were included. All volunteers signed an informed consent form before being included in the study. The study was approved by the Regional Committees for Medical and Health Research Ethics in Norway (Protocol number: REK Sør-Øst 2016/932) and performed in accordance with the declaration of Helsinki.

Blood sampling

Blood samples were collected from an antecubital vein within 24 hours prior to the start of each race (Baseline), as well as immediately after finish (Finish), and at noon of the following day (Day after). Bioengineers and medical doctors were present at all times during testing, both for the collection and handling of the samples. Whole blood samples were drawn on K2EDTA (ethylene diamine tetra acetic acid) vacutainers and refrigerated immediately after blood sampling. Serum samples were drawn in serum vacutainers containing silica particles and gel separators. These were then clotted at room temperature for 30 min before being centrifuged at 2000 g for 10 min and then refrigerated. All tubes were transported refrigerated to a certified clinical laboratory (Fürst medisinsk laboratorium, Oslo, Norway) for analysis. Only complete sets of three samples were included for analysis. Analysis were conducted of CK, NT-proBNP, Creatinine, CRP, WBC, Thrombocytes, Hb, Na+, K+, Ca2+, Mg2+, AST, ALT, T3, T4 and TSH.

Data management and statistics

All data handling, statistics and plotting was performed in R [21]. Normality for all measurements was tested with the Shapiro-Wilk Normality Test. As only CRP had normal distributed values for all measurements, all data is presented and tested with non-parametric methods to ease the reading of the article, while simultaneously maintaining the statistical strength of the findings.

Statistical tests were performed on paired data with Wilcoxon Signed Rank Tests and unpaired data with Wilcoxon Rank Sum tests to assess changes from baseline measurements. The results are given as (median [1. Quartile, 3. Quartile], p-value). Correlations were performed with Spearman’s rank correlation and presented as (R-value, p-value). Correlations were performed on all possible paired values for each correlation. Critical alpha level was set as 0.05 for all statistical tests.

Results

Samples

In total 139 subjects registered and completed baseline measurements. Due to a lack of follow up, a total of 113 subjects were included for analysis with full sets of samples (Baseline, Finish, Day after): 38 samples from the Norseman 2016, 28 from the Norseman 2018, 31 from the Norseman 2019 and 15 from the Oslo Triathlon 2018. Characteristics of included subjects are given in Table 1. Due to accident some vials was damaged during transport and excluded from the analysis. Therefore, there are differences in the total number of analyses performed for each biomarker. Complete numbers of analysis per biomarker per year are given in Appendix 1.

Table 1. Characteristics of included subjects.

Norseman Olympic distance
Female Male Female Male
n 26 72 3 12
Age (Y) 38 [34–42] 34 [34–48] 39 [33–41] 43 [37–47]
Height (cm) 168 [165–170] 180 [176–184] 168 [164–176] 182 [177–187]
Mass (kg) 61 [57–64] 78 [71–84] 61 [58–66] 73 [72–81]
BMI (m/kg2) 21.5 [20.2–22.5] 24.1 [22.8–25.7] 21.1 [21.0–21.4] 22.3 [21.0–23.8]
Times
Swim (min) 76 [64–92] 76 [68–84] 33 [33–34] 32 [26–33]
Bike (min) 447 [410–507] 418 [390–460] 85 [77–88] 73 [69–78]
Run (min) 360 [327–394] 350 [315–385] 54 [50–56] 47 [44–51]
Total (min) 902 [828–996] 874 [793–930] 180 [167–182] 158 [143–169]
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Values are median [1. Quartile, 3. Quartile]

Blood sample analysis

Clinically significant increased levels were seen at the finish line of the Norseman in WBC (12.7 [11.1–15.9] 109/L, p < 0.001), CK (2450 [1620–3950] U/L, p < 0.001) and NT-proBNP (576 [331–856] ng/L, p < 0.001). CRP had an initial small increase at the finish line after the Norseman (8 [4–19] mg/L, p < 0.001) with a clinically significant increase the following day (39 [27–56] mg/L, p < 0.001 compared to baseline, < 0.001 compared to finish line). AST had increased at the finish line (99 [74–136] U/L, p < 0.001) but it increased to even higher values the day after the race (142 [99–191] U/L, p < 0.001 compared to baseline, p < 0.05 compared to finish line). CK had significantly increased at the finish line compared to baseline, and continued to increase. It displayed the highest values the day after the races (2910 [1650–4730] U/L, p < 0.001 compared to baseline).

In comparison, after the Olympic triathlon there were also statistically significant changes, though of less clinical importance, in WBC (7.8 [6.7–9.6] 109/L, p < 0.001), CK (303 [182–393] U/L, p < 0.001) and NT-proBNP (77 [49–88] ng/L, p < 0.01) at the finish line and for CRP (2 [1–3] mg/L, p < 0.001) and AST (31 [26–41] U/L, p < 0.01) the day following the event. There were significantly lower elevations in the above measurements after the Olympic triathlon compared with values after the Norseman competitions (all p < 0.001).

We conducted a correlation analysis of the clinically significant blood samples after the Norseman races. We examined correlations among the variables, reported weekly exercise, race time, age, and body mass index (BMI). NT-proBNP at the finish line was negatively correlated with BMI (-0.35, p < 0.001). There were no significant correlations for WBC. AST and CRP the day after, and CK at the finish line, were positively correlated with each other. AST was positively correlated with CRP (0.27, p < 0.05), CK (0.82, p < 0.001), and age (0.28, p < 0.05). CRP was positively correlated with AST [0.27, p < 0.05], CK (0.43, p < 0.001), BMI (0.26, p < 0.05) and race time (0.33, p < 0.01). CK was positively correlated with AST (0.82, p < 0.001), CRP (0.43, p < 0.001), BMI (0.34, p < 0.001) and race time (0.26, p < 0.01). A visualised correlation matrix is given in Fig 1.

Fig 1. Correlations.

Fig 1

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Correlation matrix with Spearman correlations between the clinically significant elevations in blood samples: NT-proBNP, white blood cells (WBC) and Creatinine Kinase (CK) measured after finish; Aspartate Aminotransferase (AST) and C-reactive protein (CRP) measured the following day; reported weekly exercise; race time; age; and BMI. The correlation coefficient is visualised by gradient colour and shape to indicate negative correlation (red) and positive correlation (blue). Non-significant results are blank with the significance level set to p < 0.05.

Subclinical changes were also observed in Hb, Thrombocytes, K+, Ca2+, Mg2+, Creatinine, AST, ALT and T4 after the Norseman and for K+, Mg2+, Creatinine, AST and TSH after the Olympic distance. All results with statistical comparison to baseline values are given in Table 2. The percentage of samples out of reference range is given in Table 3 and individual values for WBC, CRP, AST, CK and NT-proBNP are illustrated in Fig 2. The single highest value seen in CRP, AST, ALT and CK is from the same subject and the individual was found clinically well after the race and at a control 2 weeks after the race.

Table 2. Main results.

Variable Race Baseline Finish Day after
Hb N 14.8 [14.2–15.4] 14.2 [13.5–14.9] * 14.0 [13.2–14.5] ***
(g/100 mL) O 14.8 [14.4–15.2] 14.4 [14.1–15.0] 14.4 [14–14.6]
WBC N 4.0 [3.4–4.9] 12.7 [11.1–15.9] *** 7.8 [6–10] ***
(10^09 /L) O 3.2 [2.8–3.8] 7.8 [6.7–9.6] *** 5.3 [3.9–6.3] **
Thrombocytes N 214 [197–243] 246 [227–273] ** 236 [208–255]
(10^09/L) O 191 [176–218] 235 [196–274] 202 [169–217]
CRP N 1 [1–1] 8 [4–19] *** 39 [27–56] ***
(mg/L) O 1 [1–1] 1 [1–1] 2 [1–3] ***
Na+ N 140 [139–141] 140 [138–141] 140 [139–141]
(mmol/L) O 143 [142–144] 143 [142–144] 141 [140–144]
K+ N 4.5 [4.3–4.7] 4.3 [4.1–4.7] * 4.2 [3.9–4.4] ***
(mmol/L) O 4.4 [4.2–4.6] 4.7 [4.5–5.1] ** 4.4 [4.3–4.7]
Ca2+ N 2.38 [2.32–2.43] 2.45 [2.38–2.53] *** 2.36 [2.30–2.42]
(mmol/L) O 2.44 [2.40–2.49] 2.47 [2.44–2.54] 2.38 [2.35–2.43] *
Mg2+ N 0.81 [0.77–0.85] 0.90 [0.85–0.96] *** 0.87 [0.84–0.92] ***
(mmol/L) O 0.79 [0.76–0.83] 0.74 [0.68–0.76] ** 0.82 [0.80–0.87]
Creatinine N 74 [66–82] 94 [81–106] *** 82 [73–91] ***
(umol/L) O 80 [73–82] 98 [81–108] ** 81 [74–83]
AST N 27 [24–32] 99 [74–136] *** 142 [99–191] ***
(U/L) O 22 [19–27] 29 [23–34] 31 [26–41] **
ALT N 29 [24–35] 42 [36–55] *** 53 [42–65] ***
(U/L) O 27 [23–33] 31 [24–34] 29 [23–37]
CK N 158 [119–203] 2450 [1620–3950] *** 2910 [1650–4730] ***
(U/L) O 151 [97–225] 303 [182–393] ** 531 [314–670] ***
NTproBNP N 25 [20–49] 576 [331–856] *** 230 [145–380] ***
(ng/L) O 23 [20–29] 77 [49–88] ** 63 [45–103] **
TSH N 1.6 [1.1–2.2] 2.1 [1.1–3.2] 1.5 [1.0–2.3]
(mU/L) O 2.6 [2.1–3.5] 2.2 [1.7–2.5] 1.3 [0.9–1.6] ***
T3 N 4.9 [4.5–5.3] 4.9 [4.3–5.5] 4.7 [4.3–5.2]
(pmol/L) O 5.3 [5.0–5.8] 5.1 [4.7–5.2] 5.1 [4.7–5.3]
T4 N 15.8 [14.5–17.1] 18.6 [16.3–20.3] *** 16.7 [15.6–18.2] *
(pmol/L) O 16.2 [15.6–18.2] 16.3 [15.4–19.0] 15.7 [14.8–17.2]
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Values are median [1. Quartile, 3. Quartile]. P-values were calculated with Wilcoxon Signed Rank Tests to assess changes from baseline to Finish and Day after measurements.

N, Norseman; O, Olympic triathlon; Hb, Hemoglobin; WBC, White Blood Cells; CRP, C-reactive protein; AST, Aspartate Aminotransferase; ALT, Alanine Aminotransferase; CK, Creatinine Kinase; NT-proBNP, N-terminal pro Brain Natriuretic Peptide; TSH, Thyroid Stimulating Hormone; T3, Triiodothyronine; T4, Thyroxine.

* p value < 0.05

** p value < 0.01

*** p value < 0.001

Table 3. Percentage athletes above reference values.

Variable Race Baseline Finish Day after
Hb N 3% 0% 0%
(> 17 g/100mL) O 7% 7% 0%
WBC N 0% 84% 22%
(> 10 x 10^9/L) O 0% 27% 0%
Thrombocytes N 0% 0% 0%
(> 600 x 10^9/L) O 0% 0% 0%
CRP N 1% 65% 98%
(> 5 mg/L) O 0% 0% 7%
Na+ N 0% 0% 0%
(> 145 mmol/L) O 7% 20% 7%
K+ N 6% 7% 2%
(> 5 mmol/L) O 7% 27% 7%
Ca2+ N 5% 29% 3%
(> 2.51 mmol/L) O 14% 29% 0%
Mg2+ N 2% 32% 19%
(> 0.94 mmol/L) O 0% 0% 0%
Creatinine N 0% 31% 7%
(> 105 umol/L)f O 0% 33% 0%
AST N 2% 97% 100%
(> 45 U/L)f O 0% 13% 27%
ALT N 2% 22% 34%
(> 70 U/L)f O 0% 7% 0%
CK N 6% 99% 99%
(> 400 U/L)f O 7% 27% 60%
NT-proBNP N 1% 99% 94%
(> 85 ng/L)f O 0% 23% 23%
TSH N 3% 10% 2%
(> 4 mU/L) O 7% 0% 0%
T3 N 2% 2% 3%
(> 6.5 pmol/L) O 7% 0% 0%
T4 N 0% 2% 2%
(> 23 pmol/L) O 0% 7% 0%
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N, Norseman; O, Olympic triathlon; Hb, Hemoglobin; WBC, White Blood Cells; CRP, C-reactive protein; AST, Aspartate Aminotransferase; ALT, Alanine Aminotransferase; CK, Creatinine Kinase; NT-proBNP, N-terminal pro Brain Natriuretic Peptide; TSH, Thyroid Stimulating Hormone; T3, Triiodothyronine; T4, Thyroxine.

f Different upper reference value for females. These are 90 umol/L for Creatinine, 35 U/L for AST, 45 U/L for ALT, 210 U/L for CK and 170 ng/L for NT-proBNP.

Fig 2. Individual values.

Fig 2

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Results for White blood cell count, CRP (C-reactive protein), AST (Aspartate Aminotransferase), Creatinine Kinase and NT-proBNP before the race, at the finish line and on the day after the Norseman (left) and Olympic (right) distances. Individual values are given with lines connecting each participant. Green points indicate male sex and red points indicate female sex. The green dotted line is a laboratory reference value for males; red is for females and brown indicates the same reference line for both sexes. The y-axis is identical for the Norseman and Olympic distances for each biochemical marker.

Discussion

The main finding in the present study is that the majority of biomarkers used in clinical evaluation increase above reference values after prolonged exercise events, such as the Norseman. The changes are generally much more significant after the Norseman compared to the Olympic distance. The findings of greatest clinical interest are those in WBC, CRP, AST, CK and NT-proBNP because the results would indicate pathology in the resting state in a medical examination [22–26]. This is in line with previous studies on Ironman distance triathlons [7–9].

A correlation analysis of our data shows that the increase in WBC and NT-proBNP seems to be separate phenomena, while the increase in AST and CK is strongly correlated and shows some correlation with the increase in CRP.

WBC

Measurements of WBC show that prolonged exercise is related to leukocytosis, which declined the day after the Norseman. A similar but smaller leukocytosis is also observed after the Olympic triathlon. As many as 84% of the subjects were above the laboratory reference limit for WBC at the finish line after the Norseman, while only 27% of the subjects after the Olympic distance tested that high.

Exercise induced leukocytosis is a known physiological phenomenon described as early as at the end of the 19th century [27]. Circulatory catecholamine’s are known to mobilise leukocytes from the spleen, lung and marginal zone in venules [28]. An increase in catecholamines during exercise is therefore believed to be of importance concerning exercise induced leukocytosis [29, 30]. However, exercise induced leukocytosis is also shown to occur during the infusion of non-selective beta-blockers [31]. Increased blood flow through tissue with pooled leucocytes is therefore believed to work together with humoral activation to mobilise leucocytes into the circulatory blood pool during exercise [30].

Exercise induced leukocytosis is shown to have a bimodal increase after shorter periods of exercise, with a partial initial increase at the finish, followed by a transient decrease after 15–30 min, before a final increase 90–120 minutes post exercise [32]. In a study of prolonged exercise during a 24 hour race, there was a gradually increasing leukocytosis for the first 16 hours, followed by a decrease throughout the rest of the race [33]. These different profiles of leukocytosis could explain the differences between the WBC seen after Norseman, with a race time of 10–20 hours, and that after the Olympic distance, with a race time of less than 3 hours for a majority of the athletes. The values observed after the Norseman are comparable to the values reported 3 hours after short and intense exercise protocols [18, 34], but higher than what is reported after ultra-distance marathons with a duration of 9–12 hours [17]. The different levels of leukocytosis between the Norseman and ultra-distance marathons could be related to differences in muscle activation [35]. In triathlons, the athletes use both the upper- and lower body more, compared to long distance running. Another possible explanation may be a difference in intensity, or the variation of intensity, during the races [36]. Further studies of long duration exercise events with registration of both intensity and leukocyte counts are needed to answer these questions.

CRP, AST and CK

CRP, AST and CK all showed clinically significant increases after the Norseman with, respectively, 65%, 97% and 99% of the participants found above reference values immediately after the race. CRP continued to increase the day after the race, with 98% of the participants reaching levels above reference values.

CRP is an acute phase protein originating from the liver [37] stimulated by IL-6 originating at sites of pathology [9, 38]. Pathology such as muscle damage thus elevates the circulatory pool of CRP to above reference values after 6 hours and peaks after 48 hours [37, 39, 40]. This explains the continued increase of CRP the day after the Norseman. After the Olympic distance, there was no increase directly after the race and almost no increase the following day. This is in line with other studies of shorter exercise that show little change in CRP [16, 41]. CRP after the Norseman was correlated to cell damage biomarkers (AST and CK), and both CRP and CK showed significant correlation with the race times in the Norseman. We therefore think that shorter events with exercise cause less damage and therefore a smaller stimulus to CRP production in the liver.

In contrast to this thesis, some recently published values of CRP during and after a 24 hour ultra-distance race were less then 10 mg/L [4] for all measurement times. However, the day following the Norseman we measured CRP to 39 [27 – 56] mg/L. This is comparable to recently published values from an Ironman triathlon [4]. It could be argued that there are differences in the fitness of the athletes in the 24 hour ultra-distance run and the Norseman. In our sample of athletes from the Norseman there are both professional triathletes and recreational triathletes, while the measurements in the 24 hour ultra-distance run had inclusion criteria stating that all subjects had to have participated in at least 5 marathons. A retrospect analysis of our data shows that only 2 out of 96 CRP values from the following day were beneath 10 mg/L and that the best triathletes also had higher CRP values. See Fig 3 for CRP values plotted against race times. This indicates that the form of exercise in an extreme triathlon, with use of the whole body and more varied intensity, is of importance to the CRP increase. This is possibly due to the associated cell damage in Ironman distance thritlons [9]. At the same time, the duration of exercise seems to be of importance. Further studies of both mechanical load, intensity and CRP are needed to better understand these differences in CRP values after the Norseman and 24 hour ultra-marathons.

Fig 3. Individual CRP values the day after the Norseman in relation to race times.

Fig 3

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Values for C-reactive protein (CRP) for all measured individuals. Red indicates female and blue indicates male. The black dashed line indicates 10 mg/L as referred to in the discussion.

Another interesting finding is that CRP was not correlated to the leukocytosis, but to the increase of CK and AST, which both are intracellular enzymes used as markers of cell damage [42]. This supports the idea that the exercise induced leukocytosis is a phenomenon that includes the mobilisation of white blood cells due to exercise induced humoral activation and increased blood flow rather than a phenomena due to cell damage [28, 43]. However we believe the CRP increase to be a response actual cell damage through IL-6 stimulation from activated leucocytes in damaged muscle tissue, elicited by prolonged and exhausting exercise [38].

NT-proBNP

NT-proBNP is the N-terminal bi product of the prohormone for BNP, a cardiac hormone [44]. BNP causes diuresis, vasodilatation and decreased renin and aldosterone secretion and therefore reduces the load on the heart through reduced peripheral resistance and reduction in blood volume [45]. NT-proBNP is used as a surrogate for BNP due to longer half time in plasma [46]. BNP is believed to be released due to strain in myocytes [47]. Increased levels are also associated, to a lesser degree, with hypoxia [48–50]. Hormonal regulation is furthermore demonstrated in vitro with increased secretion in the presence of angiotensin II and reduced secretion in the presence of endothelin in rat myocytes [51].

NT-proBNP has become a biomarker for detecting and managing heart failure with prognostic value, where higher values is associated with increased mortality [26]. There is no consensus for cut-off values, but values of > 450 ng/L have been proposed as diagnostic for heart failure for a population < 50 years [52]. In our study the median value for NT-proBNP at the finish line after the Norseman was 576 ng/L, with results ranging from 76 ng/L to 2837 ng/L, with 61% of the participants having values above 450 ng/L. We observed that 99% of the participants had values above the laboratory reference limits (85 ng/L for men and 170 ng/L for women). There was a large and significant difference between the Norseman and the Olympic distance. The high values measured after the Norseman are in line with reported values after Ironman Kalmar in Sweden [8]. The results from the Norseman and Ironman Kalmar, indicate that long distance triathlons elicit considerably higher values than reported after both ultra-distance running [4] and long distance swimming [53]. This is probably due to the combination of long time exercise and high intensity causing repeated strain on the cardiac myocytes.

Interestingly, our correlation analysis showed a negative correlation to BMI, indicating that a higher BMI protects against a NT-proBNP rise. A study of NT-proBNP and cut off values for heart failure show decreased values in obese patients with heart failure [54]. These findings are in line with our findings after the Norseman, with lower values of NT-proBNP in subjects with larger BMI who have all been through the same race. The reason for lower values with higher BMI is not elucidated. However, one could speculate that the heart of athletes with larger body mass is relatively smaller to their blood volume causing a smaller concentration of NT-proBNP in the blood [55, 56]. Another possibility is that the rate of secretion could be higher in larger subjects [55].

Strengths and limitations

This study presents biochemical markers from a relatively large number of participants offering valuable reference values for clinicians treating patients and medical teams working in Ironman distance competitions. Differences between the genders have not been the main aim of this article. But expected values for several biomarkers differ between males and females as well as finish times, therefore supplemental information are provided with values for both sexes [13, 57–59]. Norseman is known for cold swim temperatures [60, 61]. A limitation of our study is that we have not studied the effects of swimming in cold water may affect the biomarkers [62]. Another limitation is variation in time between finish blood samples and blood samples from the day after the race. This was caused by the large variation in finish times and that all blood samples form the day after the race were collected at noon due to practical concerns.

Conclusion

In conclusion, prolonged exercise events, such as Norseman, induces significant changes in biomarkers used in a clinical setting. Several are previously known to change during exercise, but this study describes the magnitude of increase in an Ironman triathlon competition, compared to a shorter competition, the Olympic distance triathlon. Of greatest clinical importance in the present study are the large increases in leukocytes, CRP, AST, CK and NT-proBNP after the Norseman. This is important to be aware of when athletes engaging in prolonged exercise competitions receive medical assistance or are hospitalised during or after an event like the Norseman. We publish our measured values after the Norseman as a guiding tool for expected physiological changes for clinicians treating patients and medical teams in prolonged exercise events.

Supporting information

S1 Table. Included samples.

(DOCX)

Click here for additional data file. (16.1KB, docx)
S2 Table. Results for only males.

(DOCX)

Click here for additional data file. (18.2KB, docx)
S3 Table. Results for only females.

(DOCX)

Click here for additional data file. (18.1KB, docx)
S4 Table. Differences between genders.

(DOCX)

Click here for additional data file. (16.8KB, docx)
S1 Raw data. CSV file containing raw data.

Age and race times are not included, to comply with the ethical approval of the study and Norwegian law, as this would make the subjects identifiable since results from the races are public.

(CSV)

Click here for additional data file. (24.2KB, csv)

Acknowledgments

This study was conducted by the Norseman Research team. The Norseman Research team consists of scientists from serval institutions furthering the knowledge of extreme endurance exercise in cooperation with the Norseman Xtreme Triathlon and volunteering participants.

Data Availability

All relevant data are within the manuscript and its Supporting Information files. Rawdata for age and race times are not included to ensure anonymity for the participants since the results from the races are public.

Funding Statement

This study was founded through a grant form Aker BioMarine Antarctic AS (http://www.akerbiomarine.com/). The funder provided support in the form of salaries for author ABS and research materials but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the author are articulated in the ‘author contributions’ section.”

References

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Decision Letter 0

Pedro Tauler

8 Jun 2020

PONE-D-20-13546

High CRP, AST, CK, NT-proBNP and leukocytosis after the Norseman Extreme Triathlon (NXTRI)

PLOS ONE

Dear Dr. Nyborg,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please consider carefully all coments form the reviewers. It seems that ther is a general lck of proper references along the manuscript as well as spelling or grammatical mistakes. Furthermore, more details regarding methodology are required.

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: General comment

This is an interesting study on a popular sport, where the authors use correctly scientific methods. I would be happy to recommend it for publication once the authors address a few issues. My major concern is that the English and writing style must be improved throughout the text to meet the standards of PLOS ONE as in its current form some parts are hard to be understood.

Specific comments

1. Title: Adopt a more ‘neutral’ title, e.g. ‘the effect of … on…’ and delete (NXTRI)

2. l.19-24: Reduce this part in 2-3 lines.

3. l.25-30: Increase the information on participants and decrease the information on biomarkers (they are reported in l.31-40)

4. l.31-40: This part must be rewritten as it is hard to follow.

5. l.41: Add a conclusion in 2-3 lines with a practical perspective.

6. l.44: Major literature is missing (DOI: 10.4077/CJP.2016.BAE420).

7. l.49: Changes meters to m.

8. l.45-49: Add references to support the provided information.

9. l.50: The term ‘extreme exercise’ -here and throughout the text- must be revised as it is not precise. Is it prolonged? High-intensity? Extreme cold? Cold water? Large elevations? Eg the ‘prolonged strenuous’ used in l.59 is more precise.

10. l.50-58: Add 3-4 references.

11. l.61: Revise ‘extreme triathlon’.

12. l.61: The gap of the existed literature has not been identified correctly. It needs 3-4 sentences (with references) presenting what is known and what is missing.

13. l.67: Add hypotheses.

14. Introduction: Make this part more specific to the aims to the paper. All biomarkers presented in the methods and results must be introduced here.

15. l.72: When did they receive the email?

16. l.79: …noon of…

17. l.95-96: Delete references 6-13.

18. l.107: Delete ‘significant… 0.001’. Report all p values at 3 decimals, e.g. p=0.364, p<0.001…

19. l.108: Add effect size.

20. l.297: Add a paragraph showing limitations, strength and practical applications.

21. l. 297: Major literature is missing in the discussion, too (DOI 10.3389/fphys.2017.00638; 10.3389/fphys.2018.01959)

Reviewer #2: GENERAL

Thank you for sending me this interesting manuscript in which the authors have described changes in various biomarkers in response to the Norseman Extreme Triathlon. The aim of the study is to elucidate typical changes in these biomarkers, which might assist in risk stratification. The study is simple, with a straight-forward protocol. It provides useful data for Race Directors and medics who might be overseeing the event. I have no major issues with study or analyses, but more in the general write-up and reporting of Methods and Results. Important details are missing from the Methods, and sections need clarifying to help with interpreting the Results. For example, no data has been provided on the precise timescale of post-race blood samples, or in environmental conditions between Norseman and Oslo Olympic. These omissions undermine your interesting results. There are also numerous errors in language and in reporting of Results which may obscure the translation of the data. The study may deserve publication, but the manuscript needs to be considerably revised. Please see Specific comments.

SPECIFIC

Abstract

◦ Line 19-20. “Little is known about the physiological impact of such events on the human body”. I disagree with the statement; there are hundreds of studies looking at physiological responses to ultra- and extreme-endurance competition. Perhaps you could say ‘physiological impact of extreme triathlon’?

◦ Line 21. It seemed to me, from the Introduction and Discussion, that the main aim was to characterize the biomarker response, and that comparison to Olympic was a secondary aim for context. Consider re-ordering the abstract to reflect the primary and secondary aims.

◦ Line 24. Use of the word ‘injured’ here might not be appropriate; implies musculoskeletal injury.

◦ Line 25. Intravenous samples? Many of these biomarkers can be assessed with pin-prick capillary.

◦ Line 31. “Increased levels of clinical significance”. Just say ‘significant increases’.

◦ Line 33. “Clinically significant changes”. Changes could be increase or decrease, be specific.

◦ See my comment below in Results; the post-race absolute values need context. The absolute vealues presented here don't mean anything. Why not express the Results as percentage increase from baseline? This way you provide context against baseline values.

◦ Abstract needs a concluding statement. How might the data be used?

Keywords. These contain several misspelled words, e.g., ‘Extreeme’ and ‘Thriathlon’. Also, keywords are generally those not included in the title. Reconsider.

Title. The title is not descriptive, and also contains technical abbreviations. Please amend the title congruent with PLOS ONE submission guidelines (https://journals.plos.org/plosone/s/submission-guidelines). Titles should be “Specific, descriptive, concise, and comprehensible to readers outside the field. Avoid specialist abbreviations if possible”. This should have been highlighted by the journal before being sent for review.

Introduction

◦ Line 45. Given that the citations are non-academic resources, please caveat that Norseman has been rated by commercial/media/popular outlets as one of the world’s toughest triathlons.

◦ Line 46. Please use SI units for meters (m), here and throughout.

◦ Line 51. Please change ‘world’ to possessive world’s.

◦ Line 61. Others have already evaluated biomarkers in response to Ironman (Danielsson et al - PMID: 28609447; Carlsson et al - PMID: 27483401; Neubauer et al - PMID: 18548269). Why haven’t these studies been mentioned here?

Line 61. I think much of the rationale for the study is based on the idea that Norseman is in a different category of triathlon. For your paper to be unique, please expand upon why Norseman is more extreme than ‘normal’ Ironman distance tri.

Methods

◦ Line 71. It would serve you to mention that Norseman and Oslo Olympic were both contested in August (i.e., similar anticipated weather); I had to look this up as the data weren’t provided. What time of day were the starts? Do you have any data on the weather conditions during both races? This would support your argument that the races were comparable in all but distance/terrain, and would lend validity to your comparisons.

◦ Line 79. Please state an approximate timescale for the first post-race blood sample; i.e., ‘within 5 min of race completion’ or ‘within 1 h of race completion’.

◦ Line 79. Why ‘noon’ the following day? Surely you didn’t collect >100 samples at noon? Be specific. How many hours on average after the finish were samples collected? Include means and STDEV. When assessing biomarkers, and in a study designed to elucidate the time-course of recovery, these details are paramount.

◦ Blood Sampling. For clarity, I would recommend integrating the first and last paragraphs (the protocol) and then following with the second paragraph (the measures). Also, I’m assuming samples were taken from an antecubital vein, but please state in the text.

◦ Line 104. No problem with the stats, but I’m confused why you’ve used Bonferroni post-hocs only on AST, CRP, and CK. Did you not assess each variable at three timepoints at both events? Sorry if I’m missing something.

◦ Line 104. I think the stats section needs a little clarity on the ‘why’. Be specific with your reasons for performing a given test, spell it out. For example, ‘to assess biomarkers among the three time-points (pre-, post-, recovery), a Wilcoxon Signed Rank Test…’. Moreover, line 105 is the first mention of correlations. State why you’re doing these: ‘to assess for associations among the variables, a Spearman’s Rank…’. Assessing associations should be in the aims.

◦ Line 104. Please capitalize ‘Spearman’s’.

◦ Line 107. To avoid repetition, consider replacing the first sentence by saying that ‘critical alpha level was set as 0.05’.

Results

◦ Line 112. Please use ‘before, after, +24h’, or ‘pre, post, post-day’, or some equivalent, but avoid mixing your time-points as you’ve done here ‘pre, after, post-day’. There needs to be consistency here for clarity. In the tables you’ve used ‘before, finish, after’. Even this is vague. What about ‘Start, Finish, +24’?

◦ Line 114. These things happen. Is this necessary information, though? i.e., do you anticipate there being differences among the years? If not, consider removing this statement.

◦ Line 116. Please clarify what is meant by ‘in some instances, single vials were discarded’. For what reason?

◦ Line 117. ‘There are differences in the total number of analyses…’. This line seems to contradict line 112 where you mentioned that ‘113 subjects were included for analysis with full sets of samples’. I think what you mean is that 113 subjects provided blood samples start, finish, and +24, but that not every measure was assessed in each participant. Again, be specific. Why were all measures not available from all subjects?

◦ Table 1. I’m sure this will be adjusted in the journal formatting, but title should always be above tables and below figures.

◦ Table1. Why provide height to 2 decimal places, but performance times and ages to none?

◦ Table 1. Consider changing ‘weight’ to ‘mass’. Also, provide mass to 1 d.p.

◦ Table 1. Age SI unit is ‘y’

◦ Interesting in itself that males / females had comparable swim times.

◦ Line 124. WBC increased to 14.2 *109/L after the race. This is different to what’s reported in Table 2. What am I missing here?

◦ Results. The post-race absolute values are meaningless if not given in the context of the baseline values. E.g., CK up at 2450 U/L doesn’t tell me about the magnitude of the increase. Seeing as you provide the raw data in Table 2, why not express the Results as percentage increase from baseline? This way you are providing something different to what I can find myself in the table, but it also provides context against baseline values.

◦ Line 139. The correlations; are these the R values? If so, please clarify in the text.

◦ Line 140. ‘Between’ variables implies only two. ‘Among’ variables means more than two.

◦ Line 154. Non-significant correlations aren’t technically correlations.

◦ Table 2. Caption, please use ‘Wilcoxon’ instead on Wilcox.

◦ Fig 2. I appreciate your desire to show individual values, but with such a large sample there is far too much data on this graph. It is difficult to read. Either use a light grey line for individual data and a thick dark line for means, or just display the means and SD/IQ.

◦ Fig 2. Given the female sample is 1/3 of the male, do you expect there to be meaningful differences among the variables? Also, is a comparison between sexes an aim of the study? If not, why present the data as two cohorts on the graphs?

◦ Fig 1/correlations. I’ve not seen correlations shown this way before. I think it’s an interesting and unique way of showing the data. Does it really add to what’s been written in the text? At your discretion.

Discussion

◦ I would like to see some more comparisons of your data against values reported in other Ironman distance triathlons. Also, there is very little mention of the factors that make Norseman so unique; e.g., the cold-water temperatures, the tough ascents/descents (presumably greater muscle damage with downhill components in running). How might these factors influence your measured variables. This is important to distinguish your study from others looking at ‘normal’ Ironman.

◦ Line 200. ’Measurements of WBC show that extreme exercise is related leukocytosis’; please check, is the word ‘to’ missing here?

◦ Line 205. Needs a citation.

◦ Line 209. You have switched from US to UK spelling of leukocyte/leucocyte. Be consistent.

◦ Line 221. Could the difference be due to the relatively greater stress in ultra-marathon due to impact forces?

◦ Line 223. Do you mean that IL6 production elevates CRP, or is CRP produced by CRP in a positive feedback mechanism?

◦ Line 250. The 24 h race you cite comprised loops of a flat course, so I could believe that exercise intensity and/or strain was greater in Norseman, but I’m not sure about this for many/most 24-hour races performed on trails, mountains, in heat, etc. Could other conditions like the cold water have influenced the values?

◦ Line 263. Swap the word ‘thesis’ for ‘notion’ or ‘idea’.

◦ Line 289-290. Possibly the result of slower races times and, therefore, lower intensity? Although I see you found no correlation here between race time and BMI, which is unusual.

Reviewer #3: Line 22: the aim of the study was

Line 26: explain that n=98 was for 3 years

Lines 45-49: I suggest adding references such as Chin J Physiol. 2016 Oct 31;59(5):276-283. doi: 10.4077/CJP.2016.BAE420 or Springerplus. 2015 Sep 2;4:469. doi: 10.1186/s40064-015-1255-5

Line 50: add a reference

Lines 52-53: add a reference

Lines 60-61: add a reference

Line 61: the aim of the study was

Line 67: what is the hypothesis of your study?

Line 69: what were the criteria of inclusion/exclusion to the study?

Line 200: is related to

Lines 204-205: add a reference

Lines 206-207: add a reference

Lines 207-208: add a reference

Lines 220-221: add a reference

Lines 221-222: add a reference

Lines 222-223: add a reference

Line 232: add a reference

Lines 232-233: add a reference

Lines 250-252: add a reference

Lines 252-253: add a reference

Line 261: was not related to

Line 261-262: add a reference

Lines 262-265: add a reference

Lines 265-266: add a reference

Line 269: add a reference

Lines 269-270: add a reference

Lines 270-271: add a reference

Lines 271-271: add a reference

Lines 284-285: incomplete sentence

Line 289: showed a negative

Line 289: indicating that a higher

Lines 293-295: add a reference

Lines 295-296: add a reference

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PLoS One. 2020 Sep 23;15(9):e0239158. doi: 10.1371/journal.pone.0239158.r002

Author response to Decision Letter 0

17 Aug 2020

Response to Reviewer #1

• We have changed to a more ‘neutral’ title.

• The abstract is revised based on your and the other reviewers input.

• Thanks for relevant major literature. This is included in the introduction and discussion.

• Changes meters to m in the characteristics table.

• References are added were requested.

• The term “extreme exercise” is revised to “prolonged exercise” as suggested.

• The need for reference values for medical teams is the main gap in literature and is described.

• The participants received e-mails 1-2 months prior to the events. This is included in the methods chapter.

• Hypotheses and aim has been clarified in the introduction.

• The introduction has been simplified and made more specific.

• Sugested grammatical changes have been altered

• A paragraph with strengths and limitations has been added. Practical applications have been clarified in the introduction and conclusion.

Response to Reviewer #2

• The concerns about time between measurements and the issue with swimming in cold water are addressed in a new paragraph with strengths and limitations.

• We agree that much is known about the physiological impact of prolonged exercise and have reformulated our introduction. We have also included the major literature you have made us aware of.

• The word “injury” is removed due to revision of the abstract. The word accident is used in the introduction, as this is a major concern in large scale Ironman races as Norseman.

• We did intravenous blood samples form the antecubal vein. This is clarified in the method section.

• We have used the word “changes” as some of the measured electrolytes could potentially also shown decreased values. However all clinically significant changes that we found where increased values.

• We have chosen to keep the absolute values in the results chapter. The reasons are that the aim of our article is to provide reference values for clinicians and therefore the absolute value is our primary goal. As well as all of these biomarkers and their values should be familiar for medical personnel and it is absolute values that are used in the clinic, not relative change. However the relative change can easily bed deduced from the tables and the raw data if warranted.

• The practical usage of the data is addressed.

• SI units have been corrected. We chose to change the scale to cm due to a rounding-off issue addressed in the review process.

• We did not find any major changes between other papers describing biomarkers after Ironman triathlons.

• The statistics section is simplified and revised according to input from you and other reviewers.

• We changed to utilize “Baseline”, “Finish” and “Day After” throughout the article to simplify the reading of the time points.

• It was R-values in the correlations; this is clarified in the statistics section.

• You spotted a honest mistake about the value of the WBC at finish line. By mistake it was switched with the Hb value. This is corrected.

• We decided to keep individual connected values in Figure 2. We also would like to keep separation of the genders by color. We don’t believe gender differences to be a major concern in the article, but there are some differences. This is addressed in the new strengths and limitation section and made available to study through the supplemental information provided. There are also different reference values between the sexes for some of the biomarkers. Therefore we would like to fully visualize the data for readers familiar with the biomarkers and these gender differences (e.g. NT-proBNP) so they can be evaluated. Connected individual values give both the size of measure, spread and the direction of change (the main parameter in most statistical tests). So we believe this figure gives valuable information to certain readers.

• It can often be hard to understand the clustered connection in multiple correlations. We therefore believe the visualized matrix give valuable insight. While the text give exact coefficients and p-values.

• We have uniformed the use of leukocyte/leucocyte.

• The text concerning IL-6 and CRP is clarified. IL-6 is produced by leucocytes at site of pathology. IL-6 then stimulates CRP production in the liver.

Response to Reviewer #3

• Major literature that you provided is now included.

• Hypothesis and aim is clarified.

• The inclusion criteria are clarified. We had no exclusion criteria.

• References are provided were warranted.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (20.9KB, docx)

Decision Letter 1

Pedro Tauler

1 Sep 2020

Biochemical markers after the Norseman Extreme Triathlon

PONE-D-20-13546R1

Dear Dr. Nyborg,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Pedro Tauler, Ph.D.

Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: no commentno commentno commentno commentno commentno commentno commentno commentno commentno comment

Reviewer #3: The authors have addresssed all my comments and improved the manuscript accordingly which is now ready to be accepted for publication

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Acceptance letter

Pedro Tauler

10 Sep 2020

PONE-D-20-13546R1

Biochemical markers after the Norseman Extreme Triathlon

Dear Dr. Nyborg:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Included samples.

    (DOCX)

    Click here for additional data file. (16.1KB, docx)
    S2 Table. Results for only males.

    (DOCX)

    Click here for additional data file. (18.2KB, docx)
    S3 Table. Results for only females.

    (DOCX)

    Click here for additional data file. (18.1KB, docx)
    S4 Table. Differences between genders.

    (DOCX)

    Click here for additional data file. (16.8KB, docx)
    S1 Raw data. CSV file containing raw data.

    Age and race times are not included, to comply with the ethical approval of the study and Norwegian law, as this would make the subjects identifiable since results from the races are public.

    (CSV)

    Click here for additional data file. (24.2KB, csv)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (20.9KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files. Rawdata for age and race times are not included to ensure anonymity for the participants since the results from the races are public.

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