The luteal phase of the menstrual cycle is accompanied by diminished vagally mediated heart rate variability (vmHRV). VmHRV is consistently linked to anxiety, a commonly experienced symptom during the luteal phase. However, fear conditioning, a laboratory model of anxiety, has received limited attention in the context of menstrual cycle fluctuations. This study therefore aims to explore the influence of menstrual cycle phases on instructed fear conditioning and its interactions with vmHRV.In this study, 58 healthy individuals with regular menstrual cycles, currently in the luteal or follicular phase, participated in a fear conditioning paradigm. During this experiment, two geometric figures were either paired (CS+) or not paired (CS-) with an electric shock. Linear mixed models were used to analyze the modulatory effects of the menstrual cycle phase on the startle magnitude and skin conductance responses (SCRs) to these conditioned stimuli.Results revealed higher fear differentiation (CS+ vs. CS-) during the luteal phase in the startle magnitude, driven by a startle potentiation to the conditioned stimulus (CS+). In terms of SCR, interacting effects with vmHRV revealed that individuals with high vmHRV exhibited a similar increased fear differentiation during the luteal phase, while low vmHRV individuals showed less fear differentiation. These findings suggest that during the luteal phase, individuals exhibit stronger fear-related differentiation, a pattern that is partly modulated by vmHRV. These insights shed light on potential origins of varying symptom experiences like increased anxiety during the luteal phase. However, further research is required to investigate associations between these fluctuations and symptomatology.