Data was extracted into and processed in Matlab. Phasic heart rate responses were estimated using R-R intervals. The R-R interval is the time between successive heartbeats.

To address normal heart rate variability within and across participants, the mean of the four R-R intervals preceding the start of each trial (before presentation of fixation point) was calculated to provide a measure of pre-trial R-R interval2 (Figure 1). The changes from this pre-trial R-R interval, i.e., “heart index” (iH), were then obtained by dividing each target R-R interval by the pre-trial R-R interval + the target R-R interval. For example, the interval between the first R peak and second R peak immediately following the US, divided by the pre-trial R-R interval plus the interval of the first and second R peaks, was the first post-US iH. An iH = 0.5 would indicate no change from the pre-trial heart rate. An iH value >0.5 would indicate cardiac deceleration (larger interval length), while iH value smaller than 0.5 would indicate cardiac acceleration (smaller interval length). The absolute values of iH indicated the magnitude of the phasic heart rate changes.

The dependent variables were heart rate responses to the US/NS as well as to the cues that predicted them. The iH values were calculated for the first, second, third, and fourth R-R intervals following the US/NS presentation (interval +1, +2, +3, +4 after US/NS in Figure 2). As heart rate changes may be observed in anticipation of the stimulus presentation (Groen et al., 2007; Poli et al., 2007), the iH values were also calculated for a period before the US/NS presentation. The interval between the two R peaks immediately before the US/NS is the first pre-US/NS iH and so on (intervals −1, −2, −3, −4 before US in Figure 2). Similar procedures were followed to examine heart rate responses to cues, the iH values for the two intervals following Cue 1 and 2 were evaluated (interval +1, +2 after cue in Figure 3)3.

The mean iH values and within-subject standard errors for the first through fourth interval before US onset [separated by preceding cue type: Cue 1/CS+ (blue) and Cue 2/CS– (orange)], and the first through fourth interval after US onset [separated by the cue and US type; Food/US preceded by Cue 1/CS+ (light blue), Square/NS preceded by Cue 2/CS+ (green), and Square/NS preceded by Cue 2/CS– (yellow)] during the (A) training and (B) test phase.

The mean iH values and within-subject standard errors for the first and second interval after a cue onset [Cue 1/CS+ (blue) or Cue 2/CS– (orange)] during the (A) training and (B) test phase.

For each of the post-cue and pre-US/NS intervals, average iH values were obtained for each participant separately for two trial types: those following a Cue 1/CS+ presentation, and those following a Cue 2/CS– presentation. For each of the post-US/NS intervals, average iH values were obtained for three trial types: those following US preceded by Cue 1/CS+, those following the NS preceded by Cue 1/CS+, and those following the NS preceded by Cue 2/CS–.

Two-way repeated measures linear effects of time, US/NS and cue type on the iH values were examined using GLM. To explore the timing of the heart rate changes, one-sample tests were conducted to examine whether the iH value for each interval was significantly different from 0.5. All statistical analyses were undertaken with SPSS.

As tonic heart rate is reported to change in the presence of rewarding stimuli (Rakover and Levita, 1973; Fowles et al., 1982; Blanchard et al., 2000; Nederkoorn et al., 2000; Starcke et al., 2018), we undertook a series of exploratory analyses to test for these effects in our data. Tonic heart rate changes (bpm; beats per minute) and heart rate variability (RMSSD; the root mean square of successive differences between heart beats) during the rest period before the experimental task began and during the training and test phase were examined.

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