Purpose The objective of this study is to determine whether the typical exacerbation of symptoms in patients with chronic fatigue syndrome (CFS) after a bout of exercise differs between high-intensity interval training (HIIT) or continuous (CONT) aerobic exercise of the same duration and mechanical work. Methods Participants with specialist-diagnosed CFS performed two 20-min bouts of cycling in a randomized crossover study. The bouts were either moderate-intensity continuous (70% age-predicted HR maximum) or high-intensity interval exercise, separated by at least 2 wk. Self-report questionnaires capturing fatigue, the related symptoms, and actigraphy were collected across 2 d before and 4 d after the exercise. Comparisons between exercise bouts were made using paired sample t-tests. Results Fourteen moderately affected participants who were unable to work, but not bed bound, completed the study (nine female, 32 ± 10 yr, 67 ± 11 kg). Mechanical work was matched successfully between the exercise bouts (HIIT, 83,037, vs CONT, 83,348 J, P = 0.84). Mean HR (HIIT, 76% ± 5%, vs CONT, 73% ± 6% age-predicted HR maximum, P < 0.05) and RPE (6-20) in the legs (HIIT, 15.4 ± 1.4, vs CONT, 13.2 ± 1.2, P < 0.001) were higher for the interval compared with continuous exercise. Mean fatigue scores (0-10) were similar before each exercise challenge (HIIT, 4.5 ± 1.8, vs CONT, 4.1 ± 1.7, P = 0.43). Participants reported an increase in fatigue scores after both challenges (mean difference: HIIT, 1.0 ± 1.3, P < 0.01; CONT, 1.5 ± 0.7, P < 0.001), but these exacerbations in fatigue were not statistically or clinically different (P = 0.20). Conclusions High-intensity interval exercise did not exacerbate fatigue any more than continuous exercise of comparable workload. This finding supports evaluation of HIIT in graded exercise therapy interventions for patients with CFS.