More habitual time spent engaging in prolonged sedentary behaviours increases the risk of developing hypertension. Beat-by-beat systolic (SBPV) and diastolic blood pressure variability (DBPV) are more pronounced in persons with hypertension and may be an early manifestation of blood pressure dysregulation. We tested the hypothesis that a single bout of prolonged sitting augments very short-term SBPV and DBPV. The secondary aim was to explore sex differences in prolonged sitting-induced increases in SBPV and DBPV. Thirty-three adults (22.9±1.9 years; 17 females) completed a single, 3-hr bout of prolonged sitting with beat-by-beat arterial pressure determined at baseline, 1.5-hr, and 3-hr via finger photoplethysmography. There were no sex differences observed for baseline brachial SBP (males: 122±10 mmHg; females: 111±9 mmHg), SBPV (males: 1.87±0.63 mmHg; females: 1.51±0.38 mmHg), DBP (males: 68±6 mmHg; females: 66±8 mmHg), or DBPV (males: 1.40±0.41 mmHg; females: 1.27±0.32 mmHg) (all, p>0.41). In the pooled sample, baseline SBPV (1.68±0.54 mmHg) remained unchanged after 1.5-hr (1.80±0.60 mmHg; p=0.59), but increased after 3.0-hr (1.84±0.52 mmHg; p=0.01). This post-sitting increase was driven by males (p=0.009), with no difference observed in females (p=1.00). Similarly, baseline DBPV (1.33±0.36 mmHg) was similar after 1.5-hr (1.42±0.41 mmHg; p=0.72) but was increased at 3-hr (1.50±0.34 mmHg; p=0.02). However, no sex differences in DBPV (all, p>0.07) were observed across the time points. In young, normotensive adults, a single bout of prolonged sitting augmented beat-by-beat blood pressure variability, which may provide a link between uninterrupted sitting and the development of blood pressure dysregulation.