The redistribution of blood away from the splanchnic area is an adaptation of the body enabling perfusion of critical tissues in both physiological and pathophysiological situations. During physical exercise, reduction of the splanchnic circulation occurs to meet the increased demand for oxygen and substrates in the active muscle, thereby inducing a physiological state of hypoperfusion in the GI system 
. This study provides for the first time a comprehensive overview of the processes occurring in response to splanchnic hypoperfusion.
In this study, we demonstrate that in healthy individuals, one hour of physical exercise results in splanchnic hypoperfusion and small intestinal damage, reflected by an increase in plasma I-FABP. Interestingly, the extent of intestinal damage significantly correlated with the exercise-induced hypoperfusion and with mild permeability changes in the small intestine. The most pronounced change in splanchnic perfusion, i.e. the most obvious increase in gapg-a
, occurred within the first 10 minutes of cycling, which implies that splanchnic perfusion rapidly decreases following the onset of exercise. The speed of adaptation of the splanchnic blood flow is further demonstrated by its fast recovery after cycling, which was also most prominent during the first 10 minutes following cessation of exercise. The rapid decline in splanchnic perfusion during cycling was followed by the development of significant intestinal damage with a time lag of 20 minutes. The data show a correlation between the exercise-induced splanchnic hypoperfusion and enterocyte damage (measured 20 minutes later), which is in line with animal studies and studies performed in critically ill patients demonstrating that intestinal hypoperfusion and ischemia result in intestinal cell damage 
. The exercise-induced damage to the small intestine was measured using plasma I-FABP and I-BABP, both sensitive plasma parameters of enterocyte damage that are predominantly expressed in the upper half of the villi, where the mature enterocytes reside 
. The susceptibility of the mature enterocyte population to low-flow states and ischemic events has been explained by the counter current exchange mechanism in the villus 
, creating a constant low oxygen environment at the villus tip. Intestinal hypoperfusion deteriorates the oxygen-deprived status of the villus tip even more, resulting in rapid loss of epithelial cell membrane integrity and concomitant I-FABP and I-BABP release. The increase in both I-FABP and I-BABP indicates that exercise results in small intestinal damage in general, including the ileum. Interestingly, plasma I-FABP levels gradually decreased in the 60-minute ‘reperfusion’ period. The absence of a second peak in plasma I-FABP levels in this 60-minute period after splanchnic hypoperfusion indicates that detectable reperfusion injury did not occur in our healthy individuals. Our observations are in line with recent work on human intestinal ischemia/reperfusion (I/R) studies from our group showing that after 30 minutes of full intestinal ischemia in an isolated part of the jejunum during a Whipple procedure, plasma I-FABP levels rapidly increase, whereas during reperfusion, a gradual decrease in I-FABP levels was observed 
. During reperfusion, shedding of the damaged enterocytes prevented the jejunum from a massive I/R-induced inflammatory response 
. Taken together, these results reflect the ability of the gut to withstand episodes of partial or total deprivation of blood flow, with relatively little damage to the intestinal mucosa.
Having obtained strong indications for reduced splanchnic blood flow, we hypothesized that splanchnic outflow may be reduced during strenuous physical exercise, resulting in hepatocyte damage. Plasma levels of all assessed liver parameters (i.e. L-FABP, ALT, AST, and alpha-GST) elevated significantly after exercise. However, two other factors could be responsible for the phenomenon. First, the increase in these plasma parameters could theoretically be partly attributed to an exercise-induced reduction in plasma volume. The increases, however, largely exceeded the 10% decrease in plasma volume that has been described in athletes in more extreme settings than ours 
. Alternatively, muscle transaminase release is expected in response to (exercise-induced) muscle injury 
, while L-FABP release could be due to intestinal damage. Therefore, we additionally analyzed plasma alpha-GST, an early marker of hepatocyte injury unaffected by muscle injury, haemolysis, or intestinal injury 
. In short, the significant rise in the assessed liver parameters after exercise strongly suggests the development of mild hepatocyte damage, which is in line with García-Pagàn et al. who reported a decrease in hepatic blood flow in patients with liver cirrhosis and portal hypertension after only 10 minutes of cycling at submaximal workload 
In contrast to the intestinal and liver data, we did not observe indications for renal damage during exercise. At this stage, it is unclear whether there was no hypoperfusion in the kidneys during cycling, or whether the kidney is more resistant to physiological hypoperfusion. Previous studies support the former, showing relatively unchanged renal flow after short-term splanchnic blood redistribution 
In addition to the observed epithelial cell integrity loss, activation of the inflammatory response was shown. Inflammation is characterized by neutrophil influx and activation, resulting in the release of MPO and calprotectin, as seen in the present study. MPO plays a role in oxidative processes, while calprotectin and one of its constituents, myeloid-related protein-8 (Mrp8, S100A8), are identified as strong activators of the innate Toll-like receptor 4 signaling pathway, resulting in the expression of pro-inflammatory cytokines 
. In addition to a rise in plasma calprotectin levels, increased faecal calprotectin concentrations were found after cycling in our healthy individuals, reflecting sequestration and activation of neutrophils in the intestinal mucosa, suggesting the gut to be an additional source of inflammatory products after physical exercise. Inflammation and neutrophil influx have been described previously in the human intestine, as part of I/R injury 
, while a study by Mortensen et al., assessing femoral arterial-venous differences during knee extensor exercise, pointed towards release of calprotectin from muscle tissue during exercise 
. We consider the release of both calprotectin and MPO to be enhanced in sites of tissue injury and inflammation, including muscle tissue (during exercise), but also the gut mucosa. Loss of gut barrier function, due to cellular injury and inflammation, could result in bacterial translocation. Indeed, Jeukendrup et al. found mild endotoxaemia (5–15 pg/ml) and increased levels of C-reactive protein in the majority of participants after finishing an approximately 12 h long-distance triathlon 
. While our data did not point towards bacterial translocation, the existence of mucosal damage and inflammation in the GI tract did suggest loss of epithelial barrier function in our healthy volunteers. In line, analysis of the sugar probes in plasma using our novel HPLC/MS approach revealed increased small intestinal permeability, while these permeability changes correlated with the observed intestinal injury. Interestingly, the classical analysis of sugar probes in urine did not reveal significant changes in permeability, which might be explained by the mild and temporary nature of the increase in small intestinal permeability. In short, the absence of endotoxaemia and the mild changes in small intestinal permeability found in the current study underline the physiological basis of the exercise-induced changes in GI homeostasis.
In the current study, exercise did not significantly increase large intestinal permeability. It could be argued that the large intestine is better protected against episodes of hypoperfusion, or that blood flow in the large intestine is better maintained to prevent the translocation of bacteria and their products from the lumen to the circulation. The latter is suggested by a study of Murray et al. that found a decrease in rectal mucosal blood flow of almost 30% after acute physical stress 
, while decreases ranging from 43 to approximately 80% have been reported for splanchnic blood flow during physical exercise 
In this study, we show the development of intestinal damage due to physiological hypoperfusion. Our data imply that the healthy human small intestine can withstand transient hypoperfusion, since the observed enterocyte injury did not produce GI symptoms in our healthy individuals. However, prolonged episodes of hypoperfusion may cause more severe epithelial injury that may give rise to abdominal symptoms, as observed in endurance trained athletes 
. Furthermore, it is tempting to speculate that in individuals with hampered vascular function or decreased cardiac output, physiological conditions such as ‘regular’ physical exercise and other daily activities, will rapidly result in splanchnic hypoperfusion, ultimately leading to enterocyte damage and increased gut permeability. In patients with compromised vascular function, hypoperfusion-induced epithelial dysfunction may give rise to endotoxaemia, thereby enhancing their state of chronic inflammation, as suggested by the group of Anker for patients with chronic heart failure 
. We assume that in addition to such patients, also relatively healthy elderly experience episodes of splanchnic hypoperfusion during daily activities due to the ageing of the splanchnic vascular system.
In conclusion, our study demonstrates that a single, 60-minute bout of moderate-to-high intensity exercise is accompanied by splanchnic hypoperfusion and transient epithelial integrity loss in healthy young males, reflecting the ability of the gut to withstand to episodes of physiological stress with minor consequences.