In this, the chemometric methodology in terms of design of experiments and multivariate projections can bring a valuable contribution together with experience and knowledge related to the study itself [35]. Other issues that constantly need to be considered and optimized are standardization and quality control of sample handling and analytical characterization, as well as strategies for continuous updating of models to assure robust and reliable end results [20,36,37,38,39]. 3.2. Biological Relevance Interpreting the metabolite pattern reveals that the apparent increase in fatty acids in blood serum following

Inhibitors,research,lifescience,medical exercise could be expected and does reflect an increased lipolysis and release of fatty acids from the adipose tissue. This is stimulated by catecholamines and other stress-induced hormones during exercise [40,41]. It is known that fatty acid metabolism increases in working muscle fibers and that this is related to the intensity and duration of exercise [42] Inhibitors,research,lifescience,medical together with training and muscle glycogen state [43,44]. Inhibitors,research,lifescience,medical Of the detected amino acids, Epigenetic inhibitor molecular weight aspargine, lysine, serine, phenylalanine, methionine, arginine, ornithine, proline, histidine, allothreonine, tryptophan, as well as the branched chain amino

acids (BCAAs) valine and isoleucine, all decreased significantly (Figure 2) from pre- to post- exercise, while an increase in the level of alanine was seen at the same time. Many of these amino acids, particularly alanine, play a glucogenic role in hepatic glucose production, which does increase during exercise [45]. Thus, the release of alanine from skeletal muscle into blood may have exceeded uptake to the liver. Conversely, the decreased Inhibitors,research,lifescience,medical level of the other detected amino acids may be related to greater uptake and utilization in hepatic gluconeogenesis. As the utilization of amino acids, predominantly glutamate and the

BCAAs, Inhibitors,research,lifescience,medical increases in muscle during prolonged exercise to support the muscle ATP-synthesis, a release of glucogenic amino acids from working muscles may be less than the hepatic uptake [46,47]. In addition, the increased level of inosine detected does reflect the well characterized adenine-nucleotide these catabolism (ATP→ADP→AMP→IMP→inosine) that occurs in working muscle during strenuous exercise [48,49], and, consequently, an increased release of inosine from muscle to blood [50]. In summary, this proves that the generated models based on the detected and resolved metabolites do provide biologically relevant information, which of course is key to further application of the methodology in research, as well as for clinical applications. 4. Experimental Section 4.1. Dataset 24 healthy and regularly training male subjects (age: 25.7 ± 2.7 yr; height: 182.5 ± 7.6cm; bodyweight: 77.4 ± 8.8kg; VO2peak at 59.1 ± 7.3mL kg−1min −1) volunteered to participate in the study.