Post‐exertional malaise and delayed recovery are hallmark symptoms of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Studies on repeated cardiopulmonary exercise testing (CPET) show that previous exercise negatively affects oxygen uptake (VO₂) and power output (PO) in ME/CFS. Whether this affects arterial lactate concentrations ([La_a]) is unknown. We studied 18 female patients (18–50 years) fulfilling the Canadian Consensus Criteria for ME/CFS and 15 healthy females (18–50 years) who underwent repeated CPETs 24 h apart (CPET₁ and CPET₂) with [La_a] measured every 30th second. VO₂ at peak exercise (VO_2peak) was lower in patients than in controls on CPET₁ (P < 0.001) and decreased in patients on CPET₂ (P < 0.001). However, the difference in VO_2peak between CPETs did not differ significantly between groups. [La_a] per PO was higher in patients during both CPETs (P_interaction < 0.001), but increased in patients and decreased in controls from CPET₁ to CPET₂ (P_interaction < 0.001). Patients had lower VO₂ (P = 0.02) and PO (P = 0.002) at the gas exchange threshold (GET, the point where CO₂ production increases relative to VO₂), but relative intensity (%VO_2peak) and [La_a] at GET did not differ significantly from controls on CPET₁. Patients had a reduction in VO₂ (P = 0.02) and PO (P = 0.01) at GET on CPET₂, but no significant differences in %VO_2peak and [La_a] at GET between CPETs. Controls had no significant differences in VO₂, PO or %VO_2peak at GET between CPETs, but [La_a] at GET was reduced on CPET₂ (P = 0.008). In conclusion, previous exercise deteriorates physical performance and increases [La_a] during exercise in patients with ME/CFS while it lowers [La_a] in healthy subjects.