10.1371/journal.pone.0218920.g001 Zeljko Grubac Zeljko Grubac Nikola Sutulovic Nikola Sutulovic Anida Ademovic Anida Ademovic Milica Velimirovic Milica Velimirovic Aleksandra Rasic-Markovic Aleksandra Rasic-Markovic Djuro Macut Djuro Macut Natasa Petronijevic Natasa Petronijevic Olivera Stanojlovic Olivera Stanojlovic Dragan Hrncic Dragan Hrncic Schematic illustration of experimental design time flow. Public Library of Science 2019 progesterone Results SF rats estradiol anxiety-related behavior OFF mode fragmentation TC AC 30 min OFF mode 2019-07-03 20:05:23 Figure https://plos.figshare.com/articles/figure/Schematic_illustration_of_experimental_design_time_flow_/8636858 <p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0218920#sec006" target="_blank">Materials and Methods</a> section.</p>