This study examines the acute effect of heart rate variability (HRV) biofeedback on HRV measures during and immediately after biofeedback and during the following laboratory-induced stress. Eighteen healthy males exposed to work-related stress were randomised into an HRV biofeedback group (BIO) or a comparative group (COM). Subjects completed a modified Stroop task before (Stroop 1) and after (Stroop 2) the intervention. Both groups had similar physiological responses to stress in Stroop 1. In Stroop 2, the COM group responded similarly to the way they did to Stroop 1: respiratory frequency (RF) and heart rate (HR) increased, RMSSD and high frequency (HF) power decreased or had a tendency to decrease, while low frequency (LF) power showed no change. The BIO group responded differently in Stroop 2: while RF increased and LF power decreased, HR, RMSSD and HF power showed no change. In the BIO group, RMSSD was higher in Stroop 2 compared to Stroop 1. In conclusion, HRV biofeedback induced a short term carry-over effect during both the following rest period and laboratory-induced stress suggesting maintained HF vagal modulation in the BIO group after the intervention, and maintained LF vagal modulation in the COM group.