Brief moments of blood flow occlusion followed by reperfusion may promote enhancements in exercise performance. Thus, this study assessed the 24-h effect of post-exercise ischemic conditioning (PEIC) on exercise performance and physiological variables in trained cyclists. In a randomized, single-blind study, 28 trained cyclists (27.1 ± 1.4 years) performed a maximal incremental cycling test (MICT). The outcome measures were creatine kinase (CK), muscle soreness and perceived recovery status, heart rate, perceived exertion and power output. Immediately after the MICT, the cyclists performed 1 of the following 4 interventions: 2 sessions of 5-min occlusion/5-min reperfusion (PEIC or SHAM, 2 x 5) or 5 sessions of 2-min occlusion/2-min reperfusion (PEIC or SHAM, 5 x 2). The PEIC (50 mm Hg above the systolic blood pressure) or SHAM (20 mm Hg) treatment was applied unilaterally on alternating thighs. At 24 h after the interventions, a second MICT was performed. In all the groups, the CK levels were increased compared with the baseline (p < 0.05) after the 24-h MICT. The PEIC groups (2 x 5 and 5 x 2) felt more tired at 24 h post intervention (p < 0.05). However, both PEIC groups maintained their performance (2 x 5: p = 0.819; 5 x 2: p = 0.790), while the SHAM groups exhibited decreased performance at 24 h post intervention compared to baseline (2 x 5: p = 0.015; 5 x 2: p = 0.045). A decrease in the maximal heart rate (HR) was found only in the SHAM 2 x 5 group (p = 0.015). There were no other significant differences in the heart rate, power output or perceived exertion after 24 h compared with the baseline values for any of the interventions (p > 0.05). In conclusion, PEIC led to maintained exercise performance 24 h post intervention in trained cyclists.