The aim of this study was to verify if diagnostic lumbar puncture (DLP) in post-lumbar puncture headache (PLPH) patients is related to significant changes in cerebral blood flow which could be visualized by transcranial Doppler (TCD). Sixty-six patients were enrolled in this study. TCD was performed 24 h before DLP and repeated within 24 h after the procedure. The measurements included mean velocity (V(mean)), peak systolic velocity (V(max)), and Gosling's pulsatility index (PI), in the left and right middle cerebral artery (MCA). PLPH was observed in 21 patients (32%). No significant differences were noted in V(mean), V(max) and PI between the right and left MCAs-both before DLP and following this procedure. In patients who developed PLPH, bilateral pre-puncture values of V(mean) and V(max) were significantly higher and PI was significantly lower compared to unaffected individuals. No significant differences were observed between these groups in terms of post-puncture V(mean) and V(max), but the post-puncture PI was still significantly lower in PLPH cases. In PLPH cases, the post-puncture values of V(mean) and V(max) were significantly lower than the respective baseline parameters. A significant inverse correlation was present between PLPH severity and bilateral pre-puncture PI. In conclusion, this study revealed that higher baseline values of V(mean) and V(max) and low PI in bilateral MCAs predispose patients to PLPH.