Acute studies have consistently demonstrated small-to-medium glycaemic responses to breaking prolonged sitting, yet it is not known whether acute effects are maintained following a period of intervention or whether behavioural interventions lead to sustained benefits. A single arm, 4-week intervention with pre and post "two-arm" randomised cross-over conditions, study was conducted to investigate whether reducing prolonged sitting in free-living affects acute and chronic glucose and insulin responses. Adults aged 40-75 years living with overweight or obesity with an elevated HbA1c (5.7-7.5%) underwent four experimental conditions (two prolonged sitting [CON], two sitting with a self-paced light upright movement breaks [LUMB]) in a randomised order. One of each condition was conducted before and after the intervention. A total of 33 participants completed the study. There was no change in sitting or glucose/insulin levels over the 4-week intervention. However, glucose and insulin were reduced acutely in the LUMB conditions compared with CON (glucose [mmol/L]: CON: 5.77 [5.51; 6.02], LUMB: 5.55 [5.30; 5.81], <i>p</i> = 0.006, insulin [mIU/L]: (CON: 77.70 [61.58; 93.83], LUMB: 61.28 [51.19; 71.38], <i>p</i> =  <0.001); these responses did not change over time. In conclusion, the intervention did not lead to reduced sitting time or chronic changes to postprandial metabolism.