Our study shows that 50% of female runners who entered the study already had depleted iron stores, which increased to 71% at the end of eight weeks’ endurance running. Concentrations of hepcidin tended to be lower during long-term running training and were reduced after a recovery phase, regardless of iron status at the start of the study. We also observed increased values of the sTfR-F index and sTfR after training and recovery phase.
Iron deficiency is indeed a relevant issue to influence exercise performance among female athletes and is potentiated through physical training. Without a widely accepted criterion at hand, the prevalence ranges from 40% to more than 80% 
. However, serum ferritin levels below which athletes are considered “iron deficient” vary in different studies 
. We found that our training protocol increased the risk of depleting iron stores: 42% of runners in the group with depleted iron stores at baseline developed ID or IDA regardless of the criterion used. In all of them, the ferritin values decreased below 8 µg/L. Although it is reported that the sTfR-F index is a more reliable marker of ID and remains stable in athletes despite significant day-to-day changes in either ferritin or sTfR, it appears that the sTfR-F index did not identify early functional iron depletion, as previously reported in athletes whose serum ferritin levels were marginal but did not fall below the threshold for ID 
. Recent data show strong associations between serum hepcidin and both serum ferritin and the sTfR-F index 
.Similar association was as observed in our study, when we excluded two individuals with elevated CRP. Importantly, a high degree of intraindividual response in hepcidin concentration requires careful interpretation of findings 
Another clinically relevant finding is that a short recovery phase was insufficient to normalize ferritin values as recently reported 
. Since 64% of the runners remained at low levels of iron stores, our exercise protocol induced undesirable effect. It has to be noted that general recommendation of physical exercise as healthy behaviour to reduce several diseases may also have potential for unwanted effects. The changes of some other iron-related parameters in our study, such as sTfR, mean red cell Hb, hypochromic red cells, reticulocytes and mean reticulocyte Hb, showed increased erythropoietic activity but reflect insufficient iron for normal erythropoiesis; this was observed independently of ferritin level. It is therefore likely that a prolonged training phase would probably further deplete iron stores and could lead to IDA with a negative impact on athletes’ performance 
We found that hepcidin concentration decreased during long-term running training, regardless of iron status at the start of the study, which is not as previously reported by Karl et al 
. Importantly, they reported lower serum hepcidin concentrations in female soldiers with IDA, which is in line with significant changes of hepcidin in our participants, as almost two third of them had low ferritin level in recovery phase. However, we observed increased sTfR concentrations after training as in study previously mentioned 
. It appears that the decreased levels of hepcidin in our study could be more associated through its homeostatic regulation due to iron demand rather than due to exercise-associated inflammation as no evident inflammation was detected. A recent finding suggests that there may be a difference in activity of acute post-exercise urine hepcidin response when iron stores are compromised 
. In our study there were no differences in the serum hepcidin dynamic in female runners with depleted and normal iron stores. In the study mentioned previously 
participants had ferritin levels <35 µg/L, as in all our participants at training, which could possibly explain the non-significant difference between our groups. On the other hand we may not compare the results in a straightforward manner, since it is known that serum hepcidin does not correlate with urinary hepcidin 
. As reported in previous research 
, it appears that there is a varied individual hepcidin response (). Additional caution is needed when comparing the results across studies which have used different assays for hepcidin detection because reference ranges or units may vary considerably.
Although we did not observe changes in markers of inflammation, our results and those recently reported by Karl et al 
do not preclude the possibility that repeated bouts of high intensity exercise may provide an acute stimulus resulting in transient increases of IL-6 followed by elevated hepcidin and may influence iron disruption as observed in earlier studies 
We are aware of potential limitations. Firstly, a sample of 14 participants may not be sufficient to show potentially relevant changes in markers of iron status dynamics over time, partially due to variabilty assocaited statistical issues rather than true physiological effects. This limitation is design driven as field studies usually recruit up to 20 participants 
, with only rare exceptions 
. It also remains to be determined whether our time points were completely appropriate for evaluation of short- and long-term responses. Both these issues, however, are shared with other reports and there is an absence of straightforward guidance for clinical practice. Focusing on female athletes is physiologically driven and based on a higher prevalence of ID among them. Nonetheless, in view of emerging reports, focusing on such a group can be misleading and may be considered as potential limitation 
In conclusion, on the basis of our results and those of previous studies we can confirm the relation between decreased hepcidin and iron stores in female athletes after long-term endurance running 
. Further studies are required to elucidate the cumulative effects of repeated high-intensity exercise bouts on inflammation, hepcidin and iron status in an active population.
Our findings, together with previous reports, stress the importance of ID in female athletes. This is a multifaceted issue and awareness among athletes, trainers and medical staff is of utmost importance. Simple parameters such as serum ferritin and transferrin saturation appear sufficient to identify most of the ID in the majority of athletes, but, whether reference levels commonly accepted for general population are applicable in this study population remains controversial. In athletes, tissue iron deficiency without anemia may have a negative impact on both oxygen transport and utilization 
.Thus, for top-level athletes it may be more reasonable to apply criteria used in diseased populations in order to prevent any iron deficiency related physical exercise limitations 
. This is clinically important because both in patients 
and in athletes 
, iron supplementation improves their performance. In any case, further research in terms of sampling and established or emerging biomarkers is warranted.