Background: Sleep deprivation has been reported to affect intrinsic brain connectivity, notably in the default mode network, but studies to date have shown inconsistent effects and have largely included young participants. Aims: We aimed to investigate effects of partial sleep deprivation on intrinsic brain connectivity in young and older participants. Methods: Participants aged 20-30 (n = 30) and 65-75 (n = 23) years underwent partial sleep deprivation (3 h sleep) in a cross-over design, with two resting state functional magnetic resonance imaging (fMRI) runs in each session. We assessed intrinsic brain connectivity using independent components analysis (ICA) as well as seed-region analyses of functional connectivity, and also analysed global signal variability, regional homogeneity, and the amplitude of low-frequency fluctuations. Results: Sleep deprivation caused increased global signal variability. In contrast to previous smaller studies, sleep deprivation did not cause major changes in investigated resting state networks, nor did it cause changes in regional homogeneity. Younger participants had higher functional connectivity in most examined resting state networks, as well as higher regional homogeneity in brain areas including anterior and posterior cingulate cortex. Conclusion: We show for the first time that partial sleep deprivation caused increased global signal variability. This outcome should be examined as a potential biomarker for sleepiness using independent data.