The mechanism behind prolactin (PRL) elevation in stress-induced hyperprolactinaemia (si-HP) in humans is unclear. This study aimed to investigate the effect of psychological stress and venous puncture (VP) pain on si-HP. The study included 91 individuals (nine males, 82 females) referred for HP. Anxiety levels were assessed using the Anxiety Sensitivity Index-3 (ASI-3) and the State-Trait Anxiety Inventory (STAI-1 and STAI-2), and visual scales were used to evaluate trypanophobia, nosocomephobia, and scanxiety. Serial PRL measurements (SPM) were performed with venous cannula (VC) at 0 minutes (min), 30 min, and 60 min for the first 32 patients and with the VP method at the same intervals for the following 59 patients. si-HP was defined as PRL levels at 60 min (PRL60min) decreasing to normal limits according to gender. There were 23 HP (-), 25 HP (+), and 19 si-HP individuals, as well as 24 with macroprolactinaemia. The frequency of si-HP in the VC group (n = 7/32) was similar to that in the VP group (12/59, p=0.9). The PRL0min and PRL60min levels of the VC group were similar to those of the VP group. Trypanophobia, nosocomephobia, scanxiety, ASI-3, STAI-1, and STAI-2 scale scores were similar between the si-HP group and other groups. Trypanophobia, nosocomephobia, scanxiety, ASI-3, STAI-1 and STAI-2 scales scores were not correlated with PRL0min, or PRL60min levels. Si-HP is independent of states momentary or trait anxiety, and phobias trypanophobia, nosocomephobia and scanxiety. In this context, the relevance of the nomenclature of si-HP is controversial.