elucidating the complex processes that determine the human body 's ability to adapt to specific training stimuli is crucial to improve athletic performance in elite sports . moreover , targeted exercise - based intervention programs provide enormous potential to combat the global epidemic increase of chronic metabolic and inflammatory disorders including obesity , metabolic syndrome , and type ii diabetes . the systemic and physiological responses to exercise are complex and not well understood and involve a wide range of metabolic , immunological , and hormonal changes . hereby , the immediate systemic response to acute exercise is known to be closely dependent on the type , duration , and intensity of exercise . however , also the individuals ' training status has a major impact on the systemic response to acute exercise , indicating an adaptive response to long - term changes in whole - body repetitive exercise stimuli . there is extensive research dealing with the specific response of different exercise regimes in relation of age , gender , training status , and physical conditions including obesity and diverse chronic inflammatory disorders [ 39 ] . most of these studies focus on exercise - induced responses of selected plasma cytokines altered by acute exercise stimuli [ 1014 ] . besides their roles in mediating immunological responses , specific cytokines have recently emerged to profoundly influence diverse nonimmunological processes , including metabolic functions ( e.g. , glucose and lipid metabolism ) , repair and/or prevention of tissue damage , and skeletal muscle remodeling [ 1517 ] . these diverse actions depend on complex and still largely undeciphered crosstalk with various other metabolic and humoral biomolecules that are also released in response to physical activity . these signaling molecules can act synergistically as well as in an independent manner to meet the specific physiological and metabolic demands of the exercising body . the simultaneous detection of a wide range of plasma biomolecules by recently developed multianalyte profiling methods now offers the possibility of generating a more comprehensive picture of the complex systemic responses to exercise . in the present study , an evaluation of at least 90 plasma proteins in trained and untrained individuals at rest and in response to a standardized endurance exercise protocol was conducted . this approach offers the unique opportunity to obtain more comprehensive insights into the systemic response to acute exercise under the precondition of different training statuses . our analyzed panel included various cytokines and their soluble receptors , as well as acute - phase proteins , hormones , metabolic marker proteins , and muscle damage markers . we hypothesized that regular endurance exercise should be reflected by specific baseline plasma signatures and that acute exercise - provoked plasma variable profiles should display training status dependent response patterns . the determination of protein profiles under different preconditions will contribute to a better understanding of the complex mechanisms of systemic exercise - induced adaptation processes . these findings can be of importance for training monitoring in elite sports and in the development of exercise - based strategies for the prevention and/or therapy of chronic metabolic and inflammatory degenerative diseases . in a previous study , we analyzed skeletal muscle specimen from a subset of these individuals using an off - gel liquid chromatography tandem mass spectrometry ( lc - ms / ms ) approach . proteome profiling revealed differences in various metabolic , especially mitochondrial proteins between endurance - trained and untrained individuals under resting conditions and in response to acute exercise . in addition , a subgroup analysis of participants and variables was performed in connection with extracellular dna traps and cell - free dna evaluation in plasma . healthy male endurance - exercise - trained ( eet ) athletes as well as untrained / sedentary ( sed ) individuals were recruited based on training history and aerobic capacity . the inclusion criteria for the eet group ( n = 19 ) were regular endurance exercise training of minimum 5 hours per week for at least 5 years before participation and an individual maximal oxygen uptake ( vo2max ) > 57  ml / min / kg body weight ( bw ) . the inclusion criteria for the sed group ( n = 17 ) were less than 2 hours of unspecific sports per week and a vo2max < 47  ml / min / kg bw . all experimental procedures were approved by the local ethics committee of justus - liebig university giessen , the university hospital of tuebingen , and the university of ulm . each participant underwent a medical screening and had to sign a written consent before the experimental procedure . initially , maximum oxygen uptake was determined by cardiopulmonary exercise testing ( metalyzer ; cortex biophysics , leipzig , germany ) with an incremental protocol on a bicycle ergometer ( excalibur sport ; lode , groningen , netherlands ) to determine the vo2max including capillary blood lactate diagnostics ( biosen ; ekf diagnostics , magdeburg , germany ) to evaluate the individual performance . within one to four weeks after the performance diagnostic , the main exercise protocol was performed . after an overnight fast ( from 21.00 pm to 08.00 am ) , participants received standardized breakfast and lunch on the day of acute exercise . on the day before , blood samples were taken at rest ( called the the exercise setup comprised an acute endurance exercise protocol on a bicycle ergometer for 50  min at an intensity ( watts ) corresponding to 80% of their individual vo2max . a 10  min warm - up at 60% of their individual vo2max preceded the endurance trial . study participants were instructed to drink a defined amount of water corresponding to their body weight at two points in time during the cycling exercise protocol . at ten minutes and at three hours after the acute exercise protocol , further blood samples were collected ( called  post  and  3  h ,  resp . ) . edta blood samples were collected for whole blood cell count and preparation of plasma samples . complete blood count was accomplished by synlab lab services ( synlab medical care center kassel gmbh , kassel , germany ) . edta tubes for plasma isolation were kept on ice and centrifuged at 1600  g for 10  min within 30  min after collection ( universal 320r , hettich centrifuges , tuttlingen , germany ) . plasma supernatant was collected , immediately frozen in liquid nitrogen , and stored at 80c . quantification of 90 analytes in edta plasma samples was performed at myriad rules - based medicine ( myriad rbm , austin , usa ) using humanmap v. 1.6 of their luminex - based multiplex immunoassays as described by the vendor . only variables that could be detected in at least 80% of the plasma samples were taken into account for further statistical analysis , using a procedure for multiplex measurements [ 21 , 22 ] . this procedure resulted in the inclusion of 78 of the 90 available variables of humanmap v. 1.6 . samples of the included variables that had no detectable value were set at half of the lowest measured value for the corresponding variable . to exclude biased measures due to plasma volume shift , changes in plasma concentrations were calculated from haemoglobin and haematocrit values , and concentrations of postexercise variables were corrected as described by kraemer and brown   and accomplished previously . in addition , granulocyte - colony stimulating factor ( g - csf ) was chosen for separate analysis on the basis of recent findings in its role in exercise delayed neutrophil apoptosis and progenitor cell mobilization [ 14 , 24 ] . g - csf concentration in plasma samples was measured using a high sensitive enzyme - linked immunosorbent assay ( hselisa ) kit ( r&d systems , minneapolis , usa ) according to the manufacturers ' instructions . the concentration of g - csf was calculated using mikrowin 2000 v. 4.34 ( mikrotek labor systems , overath , germany ) . mean  standard deviation ( sd ) and median with the 1 . and 3 . quartile for all included variables can be found in supplemental table s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4851935 . physiological data were statistically analyzed using wilcoxon 's rank sum test for independent samples for differences between groups and presented as mean  sd and median with the 1 . and 3 . plasma and whole blood data were statistically analyzed using a nonparametric two - factorial analysis of variance ( anova ) for longitudinal data to assess the main effects of time and group and their interaction effects . the chosen nonparametric method allows us to analyze the data without the need of normality or data transformations . the analysis is based on the so - called relative treatment effects ( rtes ) and data are presented as rte with the corresponding 95% confidence intervals ( ci ) . briefly described , a rte has a value between 0 and 1 and indicates the probability that a measurement in one group at a given time point is larger than a value of this variable in any other combination of group and time . keeping this in mind , a rte > 0.5 means that the eet group tends to have larger values than the sed group , whereas a rte < 0.5 must be interpreted as tendency towards lower values in eet group . for more details when a significant main effect was observed , appropriate post hoc pairwise comparison for different time points ( repeated nonparametric one - factorial anova for longitudinal data with time as factor ) or between groups ( wilcoxon 's rank sum test ) was performed . correlation analysis in the eet group for plasma variables that differ significantly at baseline between groups with their corresponding vo2max was performed using spearman 's rank correlation . for all analysis a p value < 0.05 was considered to be significant . whenever necessary to account for multiple testing , holm 's correction method for p values was performed . in addition , the scores of the area under the curve with respect to the increase ( auci ) were calculated for all plasma variables and compared by student 's t - test or wilcoxon 's rank sum test when appropriate as described previously . the statistics software r , version 3.0.2 , and the packages nparld   and nparcomp , graphpad prism 5.0 ( graphpad software , la jolla , usa ) , jmp pro 10.0.2 ( sas institute , cary , usa ) , and ibm spss v. 21 ( ibm corp . , armonk , usa ) were used for statistical analysis . the acute endurance exercise at 80% vo2max in watts led to a significantly higher workload in the eet group ( p < 0.001 ) . there were no differences in lactate levels and heart rate ( hr ) between the two groups during the one - hour - lasting exercise ( table 1 ) . lactate values started to increase significantly after half of the main exercise protocol was completed and remained elevated until the end of the exercise trial , with no significant differences between groups ( p < 0.001 ) . total leukocytes and neutrophil numbers significantly increased over time until 3  h after exercise . lymphocytes and monocytes increased directly after exercise . at three hours after exercise , lymphocytes had decreased to baseline , whereas monocytes remained elevated ( table 2 ) . twenty - nine of the 78 included variables showed an effect in response to the acute exercise intervention without differences between the eet and sed group ( table 3(a ) ) . marked responses to exercise were observed for the proinflammatory and chemoattractant interleukins interleukin- ( il- ) 1 beta ( il-1 ) and il-16 directly after exercise , whereas the anti - inflammatory il-1 antagonist il-1 receptor antagonist ( il-1ra ) showed a delayed increase 3  h after exercise . matrix metalloproteinase-9 ( mmp-9 ) also increased over time , whereas its antagonist , the tissue inhibitor of metalloproteinase-1 ( timp-1 ) , showed an immediate response pattern and declined to baseline values 3  h after exercise . other inflammation - associated analytes , including the neutrophil - derived extracellular newly identified receptor for advanced glycation end - products binding protein ( en - rage ) as well as myeloperoxidase ( mpo ) and the chemokine epithelial - derived neutrophil - activating protein 78 ( ena-78 ) and the t - cell - specific protein rantes ( rantes ) , also increased markedly in response to exercise and remained elevated at the 3  h time point . we could further observe increased levels of growth factors such as brain - derived neurotrophic factor ( bdnf ) , granulocyte - colony stimulating factor ( g - csf ) , stem cell factor ( scf ) , and vascular endothelial growth factor ( vegf ) directly after exercise . g - csf , scf , and vefg remained elevated 3  h after exercise in contrast to bdnf . the same pattern applies for plasma levels of adhesive glycoproteins thrombospondin 1 and von willebrand factor ( vwf ) which also increased immediately after exercise and remained elevated . the rtes of all 29 variables that display a significant time effect are clustered in figure 2(a ) . seven plasma variables differed between groups at baseline but were not affected by the exercise intervention ( table 3(b ) ) . in the eet group , elevated levels could be observed for the protease inhibitor protein alpha-2-macroglobulin ( a2macro ) , apolipoprotein ( apo ) a - i ( apo a - i ) , a major component of high - density lipoproteins ( hdl ) , and t - helper ( th ) type 2 and hematopoietic cytokine il-5 . in contrast , significant higher baseline values in the sed group were evident for the very low density lipoprotein component apo c - iii , the chemokine macrophage - derived chemokine ( mdc ) , the acute - phase protein serum amyloid p - component ( sap ) , and the adipokine leptin . fifteen of the analyzed variables showed significant main effects for both group and time ( table 3(c ) ) . we found group differences for the chemoattractant proteins il-8 and monocyte chemotactic protein-1 ( mcp-1 ) with higher levels in the eet group . both chemokines increased directly after exercise in both groups while mcp-1 already displayed elevated baseline levels in the eet group . likewise , baseline values of the anti - inflammatory il-10 differed between groups with increased levels in the eet group . in response to exercise cytokine il-10 increased in both the eet and sed group but remained elevated only in the eet group 3  h after exercise . th2-type cytokines il-3 and il-13 revealed no post hoc time effects but differed markedly between groups with higher levels in the eet group . exercise - provoked increases in tumor necrosis factor - alpha ( tnf- ) levels could only be observed in the eet group directly in response to exercise . concordantly , plasma levels of tumor necrosis factor receptor 2 ( tnfr2 ) were significantly higher in the eet group  already at rest  and an immediate increase in response to exercise could only be observed in the eet group . the protease inhibitor protein alpha-1-antitrypsin ( aat ) was elevated in the eet group compared to the sed group with exercise dependent time effects in the sed group 3  h after exercise . we further found myoglobin , an early and sensitive marker for muscle membrane damage , to be elevated in the eet group at rest . an exercise - provoked release over time could be observed in both groups , with increasing levels at 3  h after exercise . similarly , the insulin antagonist anabolic growth hormone ( gh ) differed between groups with significantly elevated levels at rest in the eet group . gh immediately increase in response to exercise and remained elevated 3  h after exercise in both groups . in contrast , elevated levels of insulin could be detected in the sed group compared to the eet group . insulin dropped immediately after exercise in both the eet and sed group but remained decreased at 3  h after exercise only in the eet group . nine of the analyzed variables exhibited a significant interaction effect between group and time ( table 3(d ) ) . extracellular matrix proteins mmp-2 and mmp-3 revealed different time curves between eet and sed group . an effect of exercise for mmp-2 could only be observed in the eet group and mmp-3 remained elevated only in the eet group at 3  h after exercise . the increase of il-6 was more pronounced in the eet group immediately after exercise but post hoc tests revealed no significant differences between groups ( p = 0.07 ) . interaction effects could also be observed for the muscle damage markers creatine kinase mb ( ck - mb ) and heart - type fatty acid - binding protein ( fabp ) . fabp increased directly after exercise with higher levels in the eet group and remained elevated in both groups . ck - mb was significantly higher at all three sampling points in the eet group . however , a delayed increase also became apparent in the sed group at 3  h after exercise . the nonparametric analysis revealed no significant main or interaction effects of time and groups for 19 variables . rtes for significant variables with group , group and time , and interaction ( group by time ) effect for eet and sed groups ( tables 3(b)3(d ) ) are clustered in figure 2(b ) . plasma variables that differ significantly between the eet and sed group at baseline ( pre ) are displayed in table 4 . correlation analyses of baseline plasma variable values with vo2max for the eet group revealed a significant positive correlation for apo a - i ( r = 0.66 , p = 0.03 ) and a negative correlation for leptin ( r = 0.68 , p = 0.02 ) . in the present study , we compared the plasma protein profiles of endurance - exercise - trained ( eet ) and sedentary ( sed ) individuals at rest and the effects of an acute bout of intensive endurance exercise on these circulating blood variables over time in both groups . the systemic response to acute exercise relies on coordinated crosstalk of body tissues to maintain metabolic and immune homeostasis [ 3234 ] . the immune system has to preserve an adequate immune function towards exogenous pathogens while maintaining self - tolerance to endogenous components that are released during exercise from skeletal muscle and other tissues . the acute endurance exercise increased circulating levels of muscle cell damage and permeability markers such as myoglobin , ck - mb , and fabp illustrating the need for an adequate self - tolerance in response to strenuous exercise . here , an increase in muscle damage markers could be observed , especially in the eet group . key cytokines il-3 , il-5 , il-7 , and il-13 , which have the ability to skew t - cell cytokine secretion to t - helper ( th ) type 2 responses , stimulate dendritic cell differentiation , and maintain t - cell homeostasis [ 35 , 36 ] , showed significant group and time effects with higher levels in the eet group . these effects could also be observed for immunosuppressive mediators il-10 and tnfr2 [ 37 , 38 ] . these findings indicate that regular exercise provokes acute as well as long - term changes in th2-type ( il-5 , il-6 , and il-13 )   and regulatory t - cell ( treg ) ( il-10 )   cytokines with more pronounced effects in trained athletes compared to sedentary participants . despite their role in inflammation immune function and metabolic regulation are tightly interconnected when it comes to exercise - induced alterations . high il-10 levels , for example , have been reported to promote insulin sensitivity in humans and protect against insulin resistance by diminishing tnf mediated intracellular signaling in adipocytes . apo a - i , a major component of high - density lipoprotein ( hdl ) particles , has been shown to have anti - inflammatory effects on blood cells and adipocytes   but is also postulated to improve cellular energy glucose metabolism and increase glucose uptake by muscle cells . here , we could observe a positive correlation between apo a - i and the endurance capacity represented by vo2max in the eet group under resting conditions . apo a - i was not affected by acute exercise but showed a significant group effect with higher levels in the eet group . in line with this , the lc - ms / ms skeletal muscle analysis revealed elevated levels of key enzymes of the oxidative energy metabolism in the endurance - trained muscle and , therefore , the ability for enhanced muscular glucose utilization . during intense endurance exercise , glucose is the major fuel for the exercising muscle mobilized from muscle and liver glycogen stores . the observed decrease in blood insulin in response to exercise , especially in the eet group , enables the mobilization of glucose for energy expenditure . one key player in the acute metabolic signaling is the pleiotropic cytokine il-6 that is locally secreted by the contracting skeletal muscle   and found to be elevated in circulation in response to exercise . in the present study , the highest values of il-6 for both groups were measured immediately after exercise with a more pronounced effect in the eet group . il-6 has been demonstrated to provide metabolic effects , like enhanced glycogenolysis and fatty acid oxidation , which correspond to the elevated abundance of mitochondrial enzymes of the tricarboxylic acid and oxidative phosphorylation in the endurance - trained skeletal muscle   and , therefore , the possibility of utilizing more substrate for energy expenditure . il-6 knockout mice showed an impaired carbohydrate and lipid metabolism and , in addition , were insensitive towards the metabolic effects of leptin despite elevated circulating plasma leptin level . our study revealed significantly lower plasma leptin levels in the eet group but no impact of acute exercise . in addition , within the endurance - trained individuals , leptin was negatively correlated with the vo2max at baseline . a reduction in blood leptin levels has recently been implicated with both an improved endurance training status and changes in body composition . in addition to its metabolic functions , leptin is considered to have proinflammatory activity and stimulates th1-type cytokines   whereas the lower leptin levels in the eet group are in line with the abovementioned shift towards th2-type cytokines in trained individuals . in contrast , the proinflammatory cytokine tnf- and the chemokines il-8 and mcp-1 revealed group and time effects and were also increased in the eet group after exercise . in the context of exercise remarkably , the protease inhibitors aat and a2macro were found to be elevated in the eet group whereas aat also increased 3  h after exercise in the sed group . aat serves as an inhibitor of neutrophil - derived elastase   and might reflect a response to exercise - provoked neutrophilia and increased elastase release to limit its adverse effects . similarly , a2macro , a well - known inhibitor of proteinases , released by neutrophils in the inflammatory process , protects human tissue from collateral damage caused by unregulated proteolytic activity . moreover , a2macro binds to several cytokines , including tnf- , il-1 , and il-6 , and modulates their biological activity . the tnf- complex may be removed by complex formation with a2macro from circulation and the biological activity of il-1 is inhibited by a2macro . in contrast , il-6 activity has been reported to remain partly active , and il-6 is protected from degradation and available for its target cells when binding to a2macro . supportive evidence comes from the regulation of mmp-2 and mmp-3 in response to exercise with significantly higher levels in the eet group . despite their role in the degradation of extracellular matrix proteins and tissue remodeling , mmps influence inflammatory processes through their functional inactivation of cytokines and chemokines like il-1 [ 51 , 52 ] . therefore , protease inhibitors and mmps might have the potential to diminish an excessive proinflammatory reaction in response to exercise . accordingly , we suggest that the trained organism initiates long - term adaptive processes to cope with exercise - induced inflammatory and metabolic stress , thus allowing performances at higher intensities without adverse effects . a deeper understanding of exercise - induced alterations offers the possibility of prescribing customized exercise as medicine in metabolic and inflammatory disorders like obesity , metabolic syndrome , and type ii diabetes . in the present study , we could demonstrate that acute strenuous endurance exercise has differential effects on circulating blood variables , depending on the athlete 's training status . while a wide range of variables responded simultaneously to exercise in sedentary and endurance - trained individuals , we also identified plasma proteins that differ between groups in response to acute exercise . in addition , the analysis revealed plasma proteins that were not affected by acute exercise but differed between groups at baseline , indicating long - term homeostatic adaptations caused by regular endurance exercise training . as a consequence of the systemic and cellular adaptation to inflammatory and metabolic stress evoked by regular exercise , trained athletes are capable of performing at absolute higher workloads . to achieve a similar systemic burden in both groups , a standardized relative workload protocol was chosen ( indicated by similar lactate and hr values ) that allows a physiological comparison between groups . in consequence , the eet group performed at a higher absolute workload and it should be considered that postexercise plasma levels might also be affected by differences in exercise intensity of both groups . we believe that our analysis provides meaningful new insights into the effects of endurance exercise on metabolic and inflammatory signaling and homeostasis . systemic blood variables that are capable of monitoring exercise adaptation are of scientific value not only to improve athletic performance in elite sports but also to develop exercise - based intervention programs in the prevention and treatment of chronic metabolic and inflammatory diseases .
acute physical exercise and repeated exercise stimuli affect whole - body metabolic and immunologic homeostasis . the aim of this study was to determine plasma protein profiles of trained ( eet , n = 19 ) and untrained ( sed , n = 17 ) individuals at rest and in response to an acute bout of endurance exercise . participants completed a bicycle exercise test at an intensity corresponding to 80% of their vo2max . 
 plasma samples were taken before , directly after , and three hours after exercise and analyzed using multiplex immunoassays . 
 seventy - eight plasma variables were included in the final analysis . 
 twenty - nine variables displayed significant acute exercise effects in both groups . 
 seven proteins differed between groups , without being affected by acute exercise . among these a2macro and il-5 were higher in eet individuals while leptin showed elevated levels in sed individuals . 
 fifteen variables revealed group and time differences with elevated levels for il-3 , il-7 , il-10 , and tnfr2 in eet individuals . an interaction effect could be observed for nine variables including il-6 , mmp-2 , mmp-3 , and muscle damage markers . 
 the proteins that differ between groups indicate a long - term exercise effect on plasma protein concentrations . 
 these findings might be of importance in the development of exercise - based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring .