Schematic illustration of experimental design time flow.

Animals (n = 42) were acclimatized to the laboratory conditions (7 days). Adaptation to the treadmill was performed in sessions of 1h (5 min ON and 5 min OFF working regime, treadmill belt speed ON = 0.02 m/s, OFF = 0m/s) during the next 3 days. Afterwards, the sleep fragmentation protocol has been applied and animals were divided into three groups (n = 14 in each group) and subjected to one of the three protocols in the treadmill during 6h of the light period: 1) sleep fragmentation (30s OFF: 90 s ON regime, 30 sleep interruptions per hour, SF group), 2) activity control group (10min ON: 30 min OFF regime, 1.5 sleep interruptions per hour, AC group), 3) treadmill control group (constantly OFF regime, 0 sleep interruptions per hour, TC group). Upon completion of sleep fragmentation protocols one cohort of animals (n = 8 from each group) has been subjected to in vivo ethological test of anxiety-like behavior (open filed, light—dart and elevated plus maze test), while another cohort of animals (n = 6 from each group) was sacrificed for blood sampling and further in vitro ELISA determination of hormonal levels (testosterone, progesterone, estradiol, corticosterone). For further details see the Materials and Methods section.