Abstract Background The accumulation of physiological stress and the presence of inflammation disturb iron management in athletes during intense training. However, little is known about the mechanisms regulating iron levels in athletes during training periods with low training loads. In the current study, we analyzed the effect of an acute exercise on early responses of iron and iron regulatory proteins at the end of such training periods. Methods The study was performed at the end of competitive phase of training. A total of 27 trained female basketball players were included in the study after application of the inclusion/exclusion criteria. The participants performed an incremental exercise on a treadmill. Blood samples were taken before the test, immediately after exercise, and after 3 h of restitution. Parameters, such as interleukin (IL) 6, hepcidin, ferritin, transferrin, hemopexin, and lactoferrin levels, total iron-biding capacity (TIBC), unsaturated iron-biding capacity (UIBC) were determined by using appropriate biochemical tests. Results The level of iron increased significantly after exercise, and then decreased within next 3 h restitution. Except for iron levels, only TIBC levels significantly increased after exercise and decreased to baseline level during rest period. No significant changes in the levels of hepcidin, IL-6, and other proteins related to the iron homeostasis were observed. Conclusions The increases in iron level after acute exercise is short-term and transient and appear to have been insufficient to induce the acute systemic effects in rested athletes.