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Abstract The vulnerability of rice ( Oryza sativa L.) to rising global temperatures poses a major threat to food security, highlighting the importance of understanding how rice, and its different cultivars, responds to recurrent heat stress (HS). This study investigated biochemical defence responses in a time‐series experiment involving priming followed by recovery and HS in two rice cultivars with contrasting thermotolerance, Nipponbare and N22, while also considering light/dark effects. Plants were grown at 27/21°C (light/dark, 12 h photoperiod) and primed at 35/27°C for 24 h, recovered for 2 days at 27/21°C, and then subjected to a severe HS challenge at 45/35°C for 24 h. Sampling started on the pre‐priming day and occurred every 12 h, at 10 min before the onset of the dark period (dusk) and the light period (dawn). Both cultivars exhibited decreased membrane peroxidation by severe stress, which was associated with defensive responses such as proline accumulation. Ascorbate peroxidase activity increased in N22 plants under priming and severe stress at dawn compared with the pre‐priming control and with Nipponbare. In contrast, total ascorbic acid content was reduced at severe stress in both cultivars compared to the pre‐priming control. This was accompanied by a higher level of oxidation of ascorbic acid in Nipponbare plants than in N22 plants. Principal component analysis confirmed that N22 plants had more intense defensive responses at severe stress than Nipponbare, particularly at dawn. Moreover, the recovery of biochemical markers to pre‐priming conditions after priming in Nipponbare plants was not as pronounced as in N22 plants, especially at dusk. Altogether, our findings indicate that biochemical responses to heat priming, recovery, and subsequent HS varied according to cultivar thermotolerance strategies and the time of day. The data provide new insights into the biochemical basis of acquired thermotolerance in rice.