Background Guidelines recommend considering workload in interpretation of the systolic blood pressure (SBP) response to exercise, but reference values are lacking. Design This was a retrospective, consecutive cohort study. Methods From 12,976 subjects aged 18–85 years who performed a bicycle ergometer exercise test at one centre in Sweden during the years 2005–2016, we excluded those with prevalent cardiovascular disease, comorbidities, cardiac risk factors or medications. We extracted SBP, heart rate and workload (watt) from ≥ 3 time points from each test. The SBP/watt-slope and the SBP/watt-ratio at peak exercise were calculated. Age- and sex-specific mean values, standard deviations and 90th and 95th percentiles were determined. Reference equations for workload-indexed and peak SBP were derived using multiple linear regression analysis, including sex, age, workload, SBP at rest and anthropometric variables as predictors. Results A final sample of 3839 healthy subjects ( n = 1620 female) were included. While females had lower mean peak SBP than males (188 ± 24 vs 202 ± 22 mmHg, p < 0.001), workload-indexed SBP measures were markedly higher in females; SBP/watt-slope: 0.52 ± 0.21 versus 0.41 ± 0.15 mmHg/watt ( p < 0.001); peak SBP/watt-ratio: 1.35 ± 0.34 versus 0.90 ± 0.21 mmHg/watt ( p < 0.001). Age, sex, exercise capacity, resting SBP and height were significant predictors of the workload-indexed SBP parameters and were included in the reference equations. Conclusions These novel reference values can aid clinicians and exercise physiologists in interpreting the SBP response to exercise and may provide a basis for future research on the prognostic impact of exercise SBP. In females, a markedly higher SBP in relation to workload could imply a greater peripheral vascular resistance during exercise than in males.