One alternative explanation for the response of reward-omission cells on omitted-reward trials is that, following a trial in which the mouse does not receive a reward, the mouse is more anxious to begin the next trial and therefore quickly begins preparing for the next forelimb movement. If reward-omission cells were actually just ‘next trial preparation cells’, then these putative earlier motor preparations on omitted-reward trials would elicit a larger response. That these cells exhibit on average no response following rewarded trials could reflect mice choosing to wait before preparing the next trial following reward delivery compared to omitted reward. We tested two predictions of this hypothesis. First, we reasoned that if, following a rewarded trial, mice choose to initiate the next trial very quickly, putative ‘next trial preparation cells’ should exhibit increased response, as they do following omitted reward. By contrast, on rewarded trials after which mice wait before initiating the next trial, the lack of motor preparations should result in a smaller response in ‘next trial preparation cells’. Second, if mice were substantially more anxious to initiate the next trial following omitted reward, inter-trial intervals (ITIs) following omitted reward trials should be shorter compared to ITIs following rewarded trials. a–d, To test the first prediction, we leveraged natural variability in mouse behaviour to identify rewarded trials after which mice initiated the next movement very quickly and therefore had the shortest ITI (the earliest time that the robot returns to permit the mouse to initiate the next trial is 2 or 3.5 s following the previous reward, each in 3 mice). For each imaging session, we identified groups of 25 rewarded trials with the longest ITIs and those with the shortest. These two groups of rewarded trials had substantially different ITIs, indicating that their next-trial-preparatory movements varied substantially (mean ITI for the ‘short’ group was 3.6 s, for the ‘long’ group 5.8 s, n = 13 sessions). Each line in a represents one imaging session. Despite the large difference in next-trial preparations in these two groups of trials, reward-omission cells remained silent in both cases, despite robust responses on omitted-reward trials (two cells from two example mice in b, c; b is the example cell from Fig. 2b, n = 97 rewarded and 25 omitted-reward trials; for c, n = 129 rewarded and 34 omitted-reward trials). Across all 69 identified reward omission cells (d), there was no tendency for a stronger response when mice initiated the next trial quickly compared to when they waited before doing so. Thus the prediction that putative ‘next trial preparation cells’ respond to earlier next-trial preparations was not borne out. e, To test the second prediction that mice were preparing the next trial more quickly following omitted-reward trials, thereby leading to greater preparatory movements encoded by putative ‘next trial preparation cells’, we grouped ITIs according to whether they followed rewarded or omitted-reward trials within each imaging session (indicated by each line). We found no consistent difference in how long mice chose to wait before initiating the next trial following either reward or omitted-reward trials (P = 0.93 Wilcoxon signed-rank test, n = 13 imaging sessions from 6 mice). Thus, the second prediction was also not borne out. Taken together, the selective response of reward-omission cells to omitted-reward trials is more likely to be related to reward than next-trial preparations.