Performance impairment as an effect of prolonged engagement in a specific task is commonly observed. Although this is a well-known effect in everyday life, little is known about how this affects central cognitive functions such as working memory (WM) processes. In the current study, we ask how time-on-task affects WM gating processes and thus processes regulating WM maintenance and updating. To this end, we combined electroencephalography methods and recordings of the pupil diameter as an indirect of the norepinephrine (NE) system activity. Our results showed that only WM gate opening but not closing processes showed time-on-task effects. On the neurophysiological level, this was associated with modulation of dorsolateral prefrontal theta band synchronization processes, which vanished with time-on-task during WM gate opening. Interestingly, also the modulatory pattern of the NE system, as inferred using pupil diameter data, changed. At the beginning, a strong correlation of pupil diameter data and theta band synchronization processes during WM gate opening is observed. This modulatory effect vanished at the end of the experiment. The results show that time-on-task has very specific effects on WM gate opening and closing processes and suggests an important role of NE system in the time-on-task effect on WM gate opening process.