The increasing prevalence of obesity and sedentary lifestyles has led to a higher incidence of metabolic syndrome (MetS) worldwide as well as in Taiwan. Middle-aged women are at a greater risk of MetS, type 2 diabetes, and cardiovascular disease than men because they have more subcutaneous fat and larger waist circumferences compared with men with equal visceral fat levels. In this study, we investigated the effects of calorie restriction (CR) and fish oil supplementation (CRF) on middle-aged Taiwanese women with MetS. An open-label, parallel-arm, controlled trial was conducted for 12 weeks. A total of 75 eligible participants were randomly assigned to the CR or CRF group. Both the dietary intervention groups were further divided into two age groups: ≤45 and >45 years. Changes in MetS severity, inflammatory status, iron status, and red blood cell fatty acid profile were evaluated. A total of 71 participants completed the trial. Both dietary interventions significantly ameliorated MetS and improved the participants’ inflammatory status. CR significantly increased the total iron-binding capacity (TIBC) whereas CRF increased hepcidin levels in women aged >45 years. Furthermore, CRF significantly increased the n-6/n-3 and arachidonic acid/docosahexaenoic acid ratios. Both interventions improved the anthropometric and MetS characteristics, including body weight, blood glucose and triglyceride levels, and the score of the homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check index. In conclusion, the 12-week dietary interventions improved the abnormal metabolic status of middle-aged obese women. CRF was demonstrated to be more effective in ameliorating postprandial glucose level and TIBC in women aged >45 years than in those aged ≤45 years.