This study was conducted in order to analyze the effect of two different recovery modalities on classical markers of exercise-induced muscle damage (EIMD) and inflammation obtained after a simulated trail running race. We chose to compare changes in immune cell mobilisation and CRP level because they are reliable indicators of acute performance deterioration, muscle damage and/or inflammation routinely evaluated in the general population and in athletes 
. The major finding was that a single exposure to WBC significantly alleviated inflammation after a strenuous exercise run. i) Delta IL-1β was significantly suppressed 1 h after exercise following WBC, compared to the PAS condition ii) Delta IL-1ra increased 1 h and 24 h after exercise following WBC compared to PAS iii) CRP increase was strongly limited in the WBC group compared to the PAS group at 24 h and until 48 h after exercise.
Principally, trail exercise will involve substantial uphill and downhill elements. The uphill tends to result in a greater exercise intensity and hence an increased metabolic cost 
. Conversely, downhill results in a lower metabolic cost than level and uphill walking at the same absolute speed 
, but it imposes greater forces on the lower limbs 
, resulting in greater eccentric loading. These eccentric muscle actions during downhill can result in temporary EIMD, which is manifested as reduced muscle function, muscle soreness (DOMS), efflux of intramuscular enzymes, and limb swelling that may last for several days after the exercise bout 
Within the injured muscle tissue there is leukocyte infiltration and local production of various pro- and anti-inflammatory cytokines which are crucial for initiating the breakdown and the subsequent removal of damaged muscle fragments 
. As expected, the present study demonstrates that trail exercise induces a significant release and peak of IL-6 (16 fold) and IL-10 (7 fold) levels early after trail exercise compared to rest (means of both groups), followed by a rapid decrease toward pre-exercise, as demonstrated in previous studies 
. However there was no significant change in the plasma concentration of the pro-inflammatory cytokine TNF-α. This lack of change was consistent with a 42 km marathon 
and iron man race, suggesting that our population is well trained to this type of exercise 
. Moreover, the fact that the plasma level of TNF-α was not affected immediately after the trail exercise, might explain why the monocytes were also not activated by the exercise 
. It is also well established that high intensity exercise (>75%
max) is associated with significant increases in circulating leukocytes (i.e.
increases of neutrophils and falls in lymphocytes) during recovery 
. In the present study, leukocytes increase an average of 34% above resting level. This is mainly due to an increase of the neutrophils number by 64% whereas lymphocytes felt to an average of 10% Post (mean of both groups). Furthermore, as previously described 
, high plasma concentration of IL-6 induces a peak expression of IL-1ra and IL-1β 1 h after exercise, 345% and 138%, respectively (PAS group values compared to Pre values).
Consistent with previous studies, we find similarly that increased cytokines levels were related to a significant increase and peak in CRP 24 h after exercise 
. In the present study, the CRP level of the PAS group increased 6-fold 24 h after the simulated running race compare to Pre value vs. 3 fold or 31 fold in previous studies 
. However, these differences compared to the first study might be explained by the greater muscle mass mobilized by lower limb vs. elbow or the used of eccentric activation vs. concentric actions in the previous study 
. Secondly, unlike results with the second study cited 
may be explained by the difference in the type and duration of exercise leading to greater acute phase response than following trail exercise. Indeed the iron man triathlon race consisted of about 10 h of exercise (swim, bike, run) vs. only 48min trail run exercise in the present study.
The amalgamation of these damaging effects can be problematic for activity on subsequent days, and there may be a greater risk of injury due to residual soreness and perturbations in muscle function 
. This study measured the selected cytokines TNF-α, IL-6, IL-10, IL-1ra, IL-1β and CRP in well-trained athletes for up to 96 h following a trail exercise. IL-6 and IL-10 levels are not influenced by one session of WBC repeated on four consecutive days. However, contrary to previous reports suggesting that WBC exposure increased the anti-inflammatory cytokine IL-10 production 
, our results present no significant changes after 4 exposures to WBC, compared to PAS modality. Nevertheless, the different type of exercise, 3 h by day of Elite training rugby during 4 days vs.
48 min running exercise in the first day might explain this difference of result between studies. However this previous study did not utilize a control passive group as in the present study, in order to state that the increase in IL-10 is due to cryotherapy and not to the repetition of exercise itself. Moreover, they conducted the study on a more acute time line, 7 days vs. 5 days in the present study, which might lead for the difference of IL-10 response.
There is accumulating evidence in the literature that IL-1β is balanced by the release of cytokine inhibitors such as IL-1ra which restrict the magnitude and duration of the inflammatory response to exercise 
. At Post 1 h, ΔIL-1ra and ΔIL-1β from Post are up-regulated and down-regulated after a single WBC session, respectively, and ΔIL-1β remain significantly different (p<0.05) at Post 24 h when compared to values taken during control passive rest recovery (). Excepted the study of Lubkowska et al. 
that showed changes of the IL-6 and IL-1 level, after multiple WBC exposure, literature on the cytokines cascade after exercise and the influence of WBC is very sparse and do not provide related results to both IL-1 and IL-6.
WBC is not effective in modulation of leukocytes population after 4 sessions of WBC following trail exercise. This result is in accordance with a previous study, which showed no significant changes in leukocytes count after 10 sessions of WBC, applied 2 days following progressive ergocycle test until volitional exhaustion 
. In parallel to IL-1 modulation, neutrophils numbers were recovered 24 h after exercise in both groups. However to the best of our knowledge, there is no previous study related to neutrophils following exercise and WBC sessions. Published data suggest that WBC has no detrimental effect on immunological parameters, although the observation period in the present study may be too short to evaluate changes in monocytes, lymphocyte involvement and function 
A previous study presented a negligible effect of WBC on CRP 
. However, we find that a single WBC exposure suppresses the peak increase in CRP 24 h after exercise and the difference (p<0.05) of ΔCRP with PAS group initiate at 1 h until 96 h after exercise (). However, the differences in exercise type between studies as previously described might also explain the differences in results. Moreover, the lower body mass index (BMI) in the study, 21.1±1.1 kg.m−2
herein vs. 27.2±2.3 kg.m−2
for the population study in Banfi et al. (2008) 
might lead to a different impact of cold at both skin and core levels. Indeed, some studies indicate reduced cold-induced thermogenesis, due to a high level of insulation in obesity under severe cold conditions 
, and decreased autonomic responsiveness 
. Indeed, a stimulating effect of cold exposure was found to depend on the relationship between the decrease in core temperature, and the duration of exposure 
In the present study, using a single exposure in WBC is associated Post 1 h with a significant decrease (p<0.05) of the pro-inflammatory mediator IL-1β () and an increase of the anti-inflammatory cytokine IL-1ra () compared to PAS. In accordance with the present results, it was shown that prolonged cold-wet (5°C) exposure following strenuous exercise also differentially modulated cytokine production, up regulating (12±3.7%) IL-1ra production and down regulating (1.1±0.05%) IL-1β secretion 
. Moreover consistent with a previous report using cold-pack application, WBC exposure immediately after exercise had no effect on IL-6 levels and was associated with a significant decrease of IL-1β 
. In contrast, previous study associated exercise with ice application recovery showed a significant decrease (29%) in the anti-inflammatory marker IL-1ra compare to the pre-exercise value 
. The discrepancy for the differences in cytokine responses between studies is likely due to the nature of exercise and the aim of the method of cold exposure (i.e.
decrease skin temperature or core body temperature) 
. Cryotherapy exposure causes a drive to maintain core body temperature, resulting in local vasoconstriction 
. In this case, the skin temperature would be a determining factor in the shortening or relaxing rate of smooth muscle in the vessel wall 
. It has been suggested that the vasoconstriction resulting from cold exposure may result in a redistribution of blood flow away from the skin towards the muscle and core. However, data of a recent study showed that more blood was distributed to the skin in cold water 
. This suggests that colder temperatures may be associated with reduced muscle blood flow, which could provide an explanation for the benefits of cold in alleviating exercise-induced muscle damage in sports and athletic contexts 
. In addition, during a severe cold exposure, such as WBC, skin temperature decreases quickly due to vasoconstriction and direct skin cooling, most markedly in the extremities 
. Indeed, this previous study showed that skin temperature recorded in the calf was 9.04±3.78°C immediately after WBC 
. Thus WBC −110°C might induce a greater fluid shift than other method, which accelerates turn-over process.
In general, we observed an exercise induced neutrophilia in all trials (Table S2
). During recovery after WBC, circulating neutrophil counts increased by an average of 114% above baseline value, with the largest increase 1 h after exercise. In contrast, the average increase in neutrophil counts was lower during PAS (101%). In accord with the result of a previous study, acute cold stress increased significantly circulating neutrophil counts 
. In the literature, neutrophils depletion significantly impaired their angiogenic function (via the vascular endothelial growth factor (VEGF)) 
. This adaptive change (angiogenesis) is one of the physiological adaptations for the improvement of perfusion, physical performance and other health benefits 
. Thus, WBC might contribute to angiogenesis, and decrease DOMS and time of recovery.
Limited evidence suggests that cold exposure may also initiate changes in cytokine expression associated with a nonspecific acute phase reaction 
. Downstream of the change in cytokine levels, especially IL-1, we observed in this study a concomitant down-regulation of CRP when athletes used the WBC treatments. Indeed, in a previous study, the correlation between IL-1 and CRP release was stronger than that IL-6 and CRP suggesting that IL-1β is probably the more powerful stimulant of CRP release 
Contrary to previous studies, we observe a significant decrease in CRP after WBC compared to PAS, while others have indicated negligible changes after WBC or CWI 
. Nevertheless, in both studies there is no assessment 24 h post-exercise attesting a significant increase or control group to observe any significant difference. Second, for Halson et al. (2008) 
, 1 min of exposure repeated 3 times to cold temperature of 11.5°C during the CWI method seems to be limited to induce sufficient physiological changes 
The mechanism underlying the abovementioned differences in cytokine generation is not clear, but it can be argued that cold-associated modulation of cytokine production may be provoked by alterations in central hemodynamics associated with enhanced thermoregulatory demands and therefore may influence immune homeostasis in cold environments 
. Since recently the direct effect of cytokines on neuroendocrine axes has been demonstrated 
. Inflammation and immunity are under the control of many different systems, including the nervous, the endocrine and the vascular systems. Nerve endings release norepinephrine in the tissues 
. Cold-induced vasoconstriction should be related to the reflex sympathetic activity and its attendant increase in the affinity of α-adrenoceptors in the vascular wall for norepinephrine (not measured in the present study) 
. It binds α and β-adrenergic receptors expressed on immune cells. Moreover, a previous study demonstrated that norepinephrine was the only hormone that responded positively to WBC treatment (i.e. three time exposure, over one week) and that the sustained norepinephrine could have a role in pain alleviation (DOMS) 
. Thus, another hypothesis has been formulated to explain the cytokines modulation. The findings of the present study (Table S1
) are consistent with investigations indicating that adrenergic/noradrenergic mechanisms are intimately involved in the regulation of cytokines production with physical stress 
. The stimulation of β-adrenoceptors during stress attenuates excessive synthesis of pro-inflammatory cytokines (IL-1β and TNF-α), and elevates anti-inflammatory cytokines (IL-6, IL-1ra and IL-10) 
. In this context, the current observations showing that cold exposure suppressed IL-1β but stimulated IL-1ra expression, indicating that β-adrenergic mechanisms may have predominated when cold stress was preceded by exercise. This confirmed that the treatment induced an anti-inflammatory protection 
In conclusion, a unique session of WBC (3 min at −110°C) performed immediately after exercise enhanced muscular recovery by restricting the inflammatory process. These findings suggest that multiple interactions between cytokines are likely involved in the physiological response to exertional fatigue and cold may serve to limit the severity of the host inflammatory response. In this case, accordingly with our hypothesis, multiple WBC exposures can enhance recovery, by decreasing the acute phase inflammatory response after a running trail exercise, thus contributing to its beneficial role in organ protection after muscle damage. The present study suggests that soluble receptor antagonist IL-1ra increases after a single whole body cryostimulation (−110°C) and restrict the inflammatory response to exercise by decrease in the magnitude of IL-1β and CRP. In term of practical applications, data confirm that the treatment induces an anti-inflammatory protection effect, and suggest that WBC reduce the time of recovery by positive effects on immunological parameters and the regeneration process.