Introduction While two distinct processes take place during fear-conditioning, - i.e., affective learning and expectancy learning - most studies use only a single read-out of associative fear memory. In affective learning the aversive valence of the unconditioned stimulus (US) is transferred to the conditioned stimulus (CS), while in expectancy learning the CS becomes a predictor of the US on a cognitive level. According to the dual process theory of fear learning, separate systems are involved in the formation and expression of the emotional and cognitive aspects of fear learning and these processes can occur independent of each other [1]. Commonly used read-outs of associative fear learning are the fear-potentiated startle reflex, pupil dilation and US-expectancy ratings. A clear asset of the fear-potentiated startle is that it reflects the affective aspect of fear learning, while pupil dilation reflects a general arousal response [2]. This makes fear-potentiated startle the preferred measure when studying the emotional aspects of fear learning. However, in order to measure fear-potentiated startle, aversively loud acoustic probes are presented during conditioning, which might in itself exert an effect on fear learning. Methods Here we tested the effect of startle probes on fear learning by comparing brain activation, measured with functional magnetic resonance imaging (fMRI), pupil dilation and retrospective US-expectancy ratings with and without acoustic startle probes within subjects. Participants 26 volunteers participated in the experiment. 10 participants were excluded from (part of) the analyses. Remaining samples consisted of 16 participants for pupil dilation and 18 for MRI and US-expectancy. Pupil dilation and US-expectancy ratings We compared pre‐exposure pupil dilation and US-expectancy (trials 1-3) to late fear-conditioning acquisition (trials 10-16). To examine fear acquisition, we analyzed the differential increase in responding. Then, we assessed the effect of acoustic startle probes on physiological and behavioral (i.e., pupil dilation, US‐expectancy ratings) fear learning. fMRI Voxel-wise statistical tests were family-wise error rate corrected for multiple comparisons (p Results Unfortunately, fear-potentiated startle signal could not be distinguished from MRI-scanning artefacts and could therefore not be analyzed. Regardless of startle probes, fear conditioning resulted in enhanced dorsal anterior cingulate cortex, insula and ventral striatum activation. Interaction analyses showed that startle probes diminished differential pupil dilation between CS+ and CS- due to increased pupil responses to CS-. A trend significant interaction effect was observed for US-expectancy and amygdala activation. Conclusions The increased pupil response to the CS- suggests that startle probes might affect fear learning in a counterintuitive way: instead of enhancing fear learning it reduced discriminative fear learning by increasing arousal to the CS-.