The ability of positive affective states to counteract negative states engendered by routine medical procedures remains poorly studied. In laboratory rats, positive affect typically associated with rough-and-tumble play can be induced through human “hand play” – the experience of being “tickled” by a human in a manner mimicking the social interactions normally occurring during physical–social play. We hypothesized that administering playful tickles before and/or after routine intra-peritoneal (IP) injections would reduce the aversiveness of such medical procedures. Accordingly, from 32 to 41 days of age, male Sprague–Dawley rats (N = 96) were either given a daily IP saline injection (INJ), or restrained similarly but not injected (control, CON), and exposed to one of four experimental treatments: no tickling, namely, left undisturbed before and after the restraint procedure (TN), or tickled for 2 min immediately before (TB), after (TA), or before and after (TBA) the restraint procedure. Rat affective response measures included rates of 50- and 22-kHz ultrasonic vocalization (USV) (validated as indicators of positive and negative affect, respectively), as well as audible vocalization rates (indicating pain and discomfort), duration of the restraint procedure, and ease-of-handling scores. Comparing INJ with CON conditions, injections reduced 50-kHz USV during (CON: 98.99 ± 3.54 calls/min, INJ: 59.2 ± 2.42, P < 0.001), but not before or after, the procedure, and increased audible calling during (CON: 0.44 ± 0.182, INJ: 0.67 ± 0.250, P = 0.0006) the procedure. Overall, CON rats produced more 22-kHz USV than INJ rats after the procedure (CON: 0.53 ± 0.158, INJ: 0.37 ± 0.134, P ≤ 0.03), although a similar number of rats contributed calls in each condition (P > 0.05). Tickling did not affect the responses of rats to injection, specifically, but increased 50-kHz USV compared to TN during the period(s) when applied (Before – TN: 8.3 ± 1.18, TB: 150.7 ± 3.16, TA: 30.9 ± 2.19, TBA: 154.4 ± 3.04; After – TN: 12.4 ± 1.39, TB: 72.5 ± 2.59, TA: 150.5 ± 3.59, TBA: 182.6 ± 2.67, P < 0.0001), and during the restraint procedure (TN: 33.6 ± 3.45, TB: 101.1 ± 4.27, TA: 76.98 ± 4.90, TBA: 105.1 ± 3.59, P < 0.0001). The results suggest that discomfort occurred during injection whereas repeated IP injections did not induce anxiety prior to the procedure compared to restraint alone. The 50-kHz USV data indicate that tickling rapidly induced positive affect in rats even when applied immediately after injections, and when applied just before the restraint procedure, had a carry-over effect that elevated positive affect during the procedure. In sum, when mildly aversive treatments must be administered repeatedly, the current findings indicate that brief tickling is more beneficial when applied pre- than post-procedure, suggesting a way to minimize potential welfare- and behaviour-disruptive effects of routine medical procedures.