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Several challenges make it difficult to simultaneously investigate central and autonomous nervous system correlates of conditioned stimulus (CS) processing in classical conditioning paradigms. Such challenges include, for example, the discrepant requirements of electroencephalography (EEG) and electrodermal activity (EDA) recordings with regard to multiple repetitions of conditions and sufficient trial duration. Here, we propose a MultiCS conditioning set-up, in which we increased the number of CSs, decreased the number of learning trials, and used trials of short and long durations for meeting requirements of simultaneous EEG–EDA recording in a differential aversive conditioning task. Forty-eight participants underwent MultiCS conditioning, in which four neutral faces (CS+) were paired four times each with aversive electric stimulation (unconditioned stimulus) during acquisition, while four different neutral faces (CS−) remained unpaired. When comparing after relative to before learning measurements, EEG revealed an enhanced centro-posterior positivity to CS+ vs. CS− during 368–600 ms, and subjective ratings indicated CS+ to be less pleasant and more arousing than CS−. Furthermore, changes in CS valence and arousal were strong enough to bias subjective ratings when faces of CS+/CS− identity were displayed with different emotional expression (happy, angry) in a post-experimental behavioral task. In contrast to a persistent neural and evaluative CS+/CS− differentiation that sustained multiple unreinforced CS presentations, electrodermal differentiation was rapidly extinguished. Current results suggest that MultiCS conditioning provides a promising paradigm for investigating pre–post-learning changes under minimal influences of extinction and overlearning of simple stimulus features. Our data also revealed methodological pitfalls, such as the possibility of occurring artifacts when combining different acquisition systems for central and peripheral psychophysiological measures.